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Production of bacteriocins by coagulase-negative staphylococci involved in bovine mastitis
Veterinary Microbiology 106 (2005) 61–71 www.elsevier.com/locate/vetmic
Production of bacteriocins by coagulase-negative staphylococci involved in bovine mastitis Janaı´na dos Santos Nascimento a, Patricia Carlin Fagundes a, Maria Aparecida Vasconcelos de Paiva Brito b, Ka´tia Regina Netto dos Santos a, Maria do Carmo de Freire Bastos a,* a
Instituto de Microbiologia Prof. Paulo de Go´es, Departamento de Microbiologia Geral, Universidade do Brazil (UFRJ), CCS, Bloco I, sala I-1-059, Cidade Universita´ria, 21941-590 Rio de Janeiro, RJ, Brazil b EMBRAPA Gado de Leite, Juiz de Fora, MG, Brasil Received 8 June 2004; received in revised form 20 September 2004; accepted 10 October 2004
Abstract In the present study, 188 coagulase-negative Staphylococcus (CNS) strains were isolated from bovine mastitis cases from 56 different Brazilian dairy herds, located in the Southeast region of the country, and were tested for antimicrobial substance production. Twelve CNS strains (6.4%) exhibited antagonistic activity against a Corynebacterium fimi indicator strain. Most antimicrobial substances were sensitive to proteolytic enzymes suggesting that they might be bacteriocins (Bac). Amongst the CNS producers, six were identified as S. epidermidis, two as S. simulans, two as S. saprophyticus, one as S. hominis and one as S. arlettae. Plasmid profile analysis of these strains revealed the presence of at least one plasmid. The Bac+ strains presented either no or few antibiotic resistance phenotypes. Three strains were shown to produce a bacteriocin either identical or similar to aureocin A70, a bacteriocin previously isolated from an S. aureus strain isolated from food. The remaining Bac+ strains produce antimicrobial peptides that seem to be distinct from the best characterised staphylococcal bacteriocins described so far. Some of them were able to inhibit Listeria monocytogenes, an important food-borne pathogen, and several strains of Streptococcus agalactiae associated with bovine mastitis, suggesting a potential use of these bacteriocins either in the prevention or in the treatment of streptococcal mastitis. # 2004 Elsevier B.V. All rights reserved. Keywords: Coagulase-negative Staphylococcus spp.; Bacteriocin; Bovine mastitis; Cattle-bacteria
1. Introduction Bovine mastitis is an inflammation of the mammary glands usually due to a microbial infection * Corresponding author. Fax: +55 21 2560 8344. E-mail address: [email protected] (M.C.F. Bastos).
that affects milk production and quality, being one of the most significant causes of economic loss to the dairy industry (Soltys and Quinn, 1999; Sordelli et al., 2000; Riffon et al., 2001; Bradley, 2002). Although several bacterial pathogens can cause mastitis, Staphylococcus aureus is the most important causative agent (followed by Streptococcus spp.), and
0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.10.014
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once it is established in the mammary glands of the animal, it is very difficult to eradicate (Soltys and Quinn, 1999; Lammers et al., 2001). Coagulase-negative staphylococci (CNS) have been considered a minor pathogen of bovine mastitis. However, many studies have recently shown the importance of CNS infection of the bovine mammary glands (Soltys and Quinn, 1999; Zhang and Maddox, 2000; Younis et al., 2003). In the efforts to avoid mastitis, some vaccines that can reduce the severity of this illness were generated. However, these vaccines still do not control efficiently the development of mastitis (Leitner et al., 2003). On the other hand, it has been verified that the indiscriminate treatment with antibiotics, without either a technical prescription or identification tests of the pathogen, can contribute to an increased resistance of these micro-organisms, making the cure of mastitis still more difficult (Gruet et al., 2001). Moreover, it is important to stand out that these medicines are set free in milk for some days, after their administration, being able to cause problems for consumers. Thus, the identification of alternative methods for controlling this illness is essential. One of these methods could be the use of bacteriocins. Bacteriocins (Bac) are antimicrobial peptides or proteins produced by bacteria with inhibitory activity against other bacteria. These substances, specially those produced by Gram-positive bacteria, present a potential use either in food industries as biopreservatives or in the prevention and treatment of some infectious diseases, having medical and veterinary applications (Jack et al., 1995; Oliveira et al., 1998b; Sahl and Bierbaum, 1998). Amongst the bacteriocin-producer staphylococcal species, S. epidermidis is the most studied. Some of its bacteriocins are very well characterised, such as Pep5, epidermin, epicidin 280 and epilancin K7, which are classified as lantibiotics. Pep5 is a 34-amino acid peptide encoded by the structural gene pepA (Kaletta et al., 1989). Epidermin is also a well-characterised lantibiotic produced by S. epidermidis Tu¨ 3298. Its structural gene, epiA, codes for a 22-amino acid peptide (Augustin et al., 1992). Epicidin 280 is also a 30-amino acid lantibiotic, produced by S. epidermidis BN280, which has 75% of homology with Pep5 (Heidrich et al., 1998). Epilancin K7, a 31-amino acid lantibiotic produced by S. epidermidis K7, is encoded by the gene elkA (van de Kamp et al., 1995).
Our laboratory has been investigating bacteriocin production by Staphylococcus strains, which is generally associated to plasmids. Amongst the bacteriocins produced by S. aureus and studied by our group, aureocins A70 and A53 are the best characterised. Aureocin A70 is a multi-peptide, nonlantibiotic bacteriocin produced by S. aureus A70 which is encoded by an 8.0 kb-plasmid named pRJ6 (Giambiagi-deMarval et al., 1990). Aureocin A70 was the first bacteriocin described which is composed of four related small cationic peptides, which are encoded by the aurABCD operon (Netz et al., 2001). Aureocin A53 is a 51-amino acid peptide produced by S. aureus A53. It is encoded on a 10.4 kb plasmid, pRJ9 (Netz et al., 2002). As well as other antimicrobial substances, bacteriocins produced by staphylococci (staphylococcins) could be employed, in their purified form, on the control of bovine mastitis. Therefore, in the present study, in an attempt to detect new bacteriocins with potential medical and veterinary applications, we extended our studies on bacteriocin production to CNS strains, which were associated with bovine mastitis in different Brazilian dairy herds.
2. Materials and methods 2.1. Bacterial strains and culture conditions One hundred and eighty-eight coagulase-negative Staphylococcus strains involved in bovine mastitis were isolated from 56 different Brazilian dairy herds located in the Southeast region of the country, and used in the screening for bacteriocin production. Seventy-four Streptococcus agalactiae strains were also isolated from bovine mastitis cases and used as indicators. These strains were isolated according to procedures recommended by Harmon et al. (1990). S. aureus and S. epidermidis strains from previous studies (Table 1) were used either as producers or as indicators in the inhibition assays. Staphylococcus strains were grown in either TSB (Difco) or BHI (Difco), at 37 8C for 18 h. TSB was used to grow the strains for DNA isolation and BHI was used in all bacteriocin assays. The bacteria used as indicators were grown in BHI medium, except for lactic acid bacteria, that were cultivated in MRS medium (BBL)
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Table 1 Staphylococcus strains previously described and used in this study Strain
Relevant features
Reference/source
Staphylococcus aureus A53 A70 A70 Bac
Bac+ (aureocin A53), Imm+, plasmid: pRJ9 (10.4 kb) Bac+ (aureocin A70), Imm+, plasmid: pRJ6 (8.0 kb) Strain A70 cured of pRJ6
Giambiagi-deMarval et al. (1990) Giambiagi-deMarval et al. (1990) Giambiagi-deMarval et al. (1990)
Staphylococcus epidermidis Tu¨ 3298 5 5 (cured) BN280 K7
Bac+ (epidermin) Bac+ (Pep5) Bac Pep5 Bac+ (epicidin 280) Bac+ (epilancin K7)
Augustin et al. (1992) Kaletta et al. (1989) Bierbaum et al. (1994) Heidrich et al. (1998) van de Kamp et al. (1995)
Bac, bacteriocin; Imm, immunity.
and incubated at 28 8C for 48 h under microaerophilic atmosphere. Bacteria were stored in their appropriate culture medium with 40% glycerol (w/v) at 20 8C until needed. When necessary, the media were supplemented with agar at 1.5% (w/v) or 0.6% (w/v).
(1990). Lack of inhibition zones when C. fimi NCTC7547 was used as the indicator strain indicated that the antimicrobial compound had either a proteinaceous or an acidic nature. 2.5. Antibiotic resistance
2.2. Assay for antimicrobial substance production This assay was done as described previously by Giambiagi-deMarval et al. (1990). C. fimi NCTC 7547 was used as the indicator strain for production of antimicrobial substances. 2.3. Identification of the strains Only the CNS strains that exhibited antimicrobial substance production were identified, using conventional biochemical tests (Kloos and Schleifer, 1986) or, when necessary, using a commercial kit for identification (API Staph, BioMe´ rieux). S. agalactiae strains were identified on the basis of Gram staining, presence of hemolysis, negative results on tests for the presence of catalase and esculin hydrolysis, and positive results on tests for CAMP and hydrolysis of sodium hypurate. 2.4. Effects of proteolytic enzymes and of 0.2N NaOH on AMS activity
Resistance patterns were determined by disc diffusion on Mueller-Hinton agar (Oxoid), according to the National Committee for Clinical Laboratory Standards (NCCLS, 2002). The following antibiotics (Sensifar) were used: ampicillin (10 mg), cephalothin (30 mg), ciprofloxacin (5 mg), clindamycin (2 mg), chloramphenicol (30 mg), erythromycin (15 mg), gentamicin (10 mg), imipenen (10 mg), mupirocin (5 mg), oxacillin (1 mg), penicillin (10 U), rifampin (5 mg), tetracycline (30 mg) and vancomycin (30 mg). The diameters of the inhibition zones were interpreted according to the NCCLS guidelines after 24 h of incubation at 37 8C. Strains which showed resistance to b-lactamic drugs were also tested for the presence of the methicillin-resistance gene (mecA) by PCR. 2.6. Isolation of plasmid DNA Whole-cell lysates were prepared as described by Giambiagi-deMarval et al. (1990). 2.7. DNA–DNA hybridisation assays
The effects of trypsin (Sigma), proteinase K (Boehinger Mannheim), protease XXIII (Sigma) and 0.2N NaOH on AMS activity were determined by the method described by Giambiagi-deMarval et al.
Southern blots and hybridisations were performed as described by Bastos and Murphy (1988). The 5.2-kb HindIII-A fragment of pRJ6 (encompassing all genes
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required for aureocin A70 production) was used as a probe. 32P-labelled probes were prepared by the Random-Primers DNA Labelling System (Invitrogen) following the manufacturer’s recommendations.
inhibitory action of the AMS produced by the CNS strains against S. agalactiae involved in bovine mastitis was also tested using 74 indicator strains.
2.8. PCR amplification
2.10. Agarose gel electrophoresis
Total DNA was extracted by boiling, as described previously by Nunes et al. (1999). The primers used for amplification of the aurABCD operon and the genes aucA, eciA and elkA (the structural genes of aureocin A70, aureocin A53, epicidin 280 and epilancin K7, respectively) as well as for amplification of the methicillin-resistance gene (mecA) and genes orfA and orfB (presumably involved in the immunity to aureocin A70) are listed in Table 2. Each PCR reaction contained: 1 PCR reaction buffer (Invitrogen), 2.5 mM concentration of each deoxyribonucleoside triphosphate, 2.5 U of Taq-Polymerase (Invitrogen) and 50 pmol of each primer. The amplification was done in a Programmable Thermal Controller (PTC-100TM, MJ Research, USA) and the cycles were performed as described by Nunes et al. (1999). A 100bp DNA ladder (Invitrogen) was used as the molecular size marker.
Agarose gel electrophoreses were performed as described by Sambrook et al. (1989). Products amplified by PCR were analysed in 1.4% (w/v) agarose gels and the plasmid profiles were determined in 0.7% (w/v) agarose gels. The gels were stained in an aqueous solution containing ethidium bromide (0.5 mg/ml) and visualised on a UV transiluminator.
2.9. Determination of the inhibitory spectrum of each AMS+ strain To determine the inhibitory spectrum of each AMS, several strains from different species of Grampositive bacteria were tested as indicators. The
3. Results 3.1. Detection and identification of the AMS+ strains One hundred and eighty-eight coagulase-negative Staphylococcus (CNS) strains were isolated from bovine mastitis cases from 56 different Brazilian dairy herds, located in the Southeast region of the country, and were tested for AMS production. Twelve CNS strains (6.4%), belonging to six different herds, exhibited antimicrobial substance production against C. fimi (inhibition zones between 17 and 38 mm). Among the CNS producers, six were identified as S. epidermidis, two as S. simulans, two as S. saprophyticus, one as S. hominis and one as S. arlettae (Table 3).
Table 2 Primers used in this work Gene(s) to be amplified
Relevant features
Primers
Size (bp) of the fragment to be amplified
aurABCD
Aureocin A70 structural genes
525
aucA
Aureocin A53 structural gene
orfA and orfB
Immunity genes to aureocin A70
elkA
Epilancin K7 structural gene
eciA
Epicidin 280 structural gene
mecA
Methicillin-resistance gene
Forward: P4B, 50 -CCTTATAACTTCGAATGCT-30 ; reverse: P5, 50 -AATTATTAACAAGAGAAA-30 Forward: AUC1, 50 -GAAGTTGTGAAAACTATTA-30 ; reverse: AUC2, 50 -CATAAAACAAAGAGCCAAAGT-30 Forward: RJ61, 50 -GACGAGGGTATTGCATA-30 ; reverse: RJ62, 50 -CTAAGCATTCGATAAGG-30 Forward: K71, 50 -ATGAATAACTCATTATTC-30 ; reverse: K72, 50 -ATGGAA AACAAAAAAG-30 Forward: Epin1, 50 -CAGGAG GGATATATTATGG-30 ; reverse: Epin2, 50 -CAATCACTACTATTGACAATCAC-30 Forward: MRS1, 50 -TAGAAATGA CTG AACGTCCG-30 ; reverse: MRS2, 50 -TTGCGATCAATGTTACCG TAG-30
322 722 171 165 154
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Table 3 Characteristics of the bacteriocin-producer CNS strains Strains b
A70 494 2166 2167 2457 3024 3154 3299 3414 3419 3528 3576 3577
Identification
S. S. S. S. S. S. S. S. S. S. S. S. S.
aureus epidermidis epidermidis epidermidis simulans epidermidis saprophyticus simulans epidermidis hominis arlettae saprophyticus epidermidis
Sensitivity of the Bac to proteolytic enzymes Protease XXIII
Proteinase K
Trypsin
S R R S S S R R R R S S S
S S S R R S S S S R S S R
S S S R S S S S S R S S R
Resistance profile
Plasmid formsa
Emc Emc Emc Ap, Ce, Gm, Ox, Pc, Tc Emc Ap, Pc Ap, Pc
8.0 (pRJ6) >25; 15; 6.7; 5.5; 3.7; 3.2; 2.2 12; 6.2 12; 6.2 >27 15; 8.1; 4.5 15; 8 >25; 3.5 >25 4 15; 8 15; 8 >25 (2)d; 2.5; 1.5
R, resistant; S, sensitive; () sensitive to all the antibiotics tested; Ap, ampicillin; Ce, cephalothin; Em, erythromycin; Gm, gentamicin; Ox, oxacillin; Pc, penicillin; Tc, tetracycline. a Size in kb. b Strain A70 was included in these experiments as a positive control. c Intermediate resistance. d The number between parentheses indicates the presence of two plasmid bands with size larger than 25 kb.
3.2. Effects of proteolytic enzymes on AMS activity The results are also presented in Table 3. The AMS were resistant to 0.2N NaOH, discarding the possibility that the inhibition exhibited was due to organic acids produced by the producer strain during its metabolism. Except for the AMS produced by the strain 3419, the remaining AMS were sensitive to at least one proteolytic enzyme tested, indicating that these AMS present a biological active proteinaceus component in their structure, the main characteristic of a typical bacteriocin. Therefore, from hereafter, the AMS+ strains will be considered Bac+. Bacteriocins produced by strains 3154, 3528 and 3576, as well as aureocin A70, showed a high sensitivity to proteolytic enzymes, being sensitive to all enzymes tested. 3.3. Antibiotic resistance profile The Bac+ strains did not exhibit a great number of resistance markers, except for strain 3024, that showed resistance to six antibiotics (Table 3). The remaining strains were resistant to either two (ampicillin and penicillin) or none of the antibiotics tested. Strains 494, 2167, 2457 and 3154 presented intermediate resistance to erythromycin. Strains that showed
resistance to one or more b-lactamic drugs were also tested for the presence of the mecA gene, which encodes methicillin resistance. Among the strains tested, only strain 3024 showed amplification of the mecA gene (data not shown). 3.4. Plasmid profiles All strains showed at least one plasmid form (Table 3). Strains 3154 (S. saprophyticus), 3528 (S. arlettae) and 3576 (S. saprophyticus) exhibited an identical plasmid profile, and, interestingly, they carry a plasmid with a size similar to that of pRJ6 (8.0 kb), a bacteriocinogenic plasmid previously characterised by our group and that encodes aureocin A70. These three strains were isolated from the same herd. Strains 2166 and 2167 (both S. epidermidis and isolated from the same herd), also presented an identical plasmid profile. 3.5. DNA–DNA hybridisation assays Since strains 3154, 3528 and 3576 carry a plasmid with a size similar to that of pRJ6, the homology between these plasmids and pRJ6 was investigated. A strong signal was detected to the 8.0-kb plasmids
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carried by strains 3154, 3528 and 3576. No hybridisation was observed in the remaining CNS strains. 3.6. Amplification of the aurABCD and orfAB operons The presence or the absence of the aureocin A70 structural genes was also confirmed by PCR using specific primers from sequences flanking operon aurABCD. The host strain of pRJ6, S. aureus A70, was used as a positive control. Strains 3154, 3528 and 3576 had a 525-bp fragment amplified. The remaining nine strains did not present amplification of this fragment (Fig. 1). Additionally, PCR experiments using primers to amplify the genes orfA and orfB, that seem to be involved in the immunity to aureocin A70, revealed that only strains 3154, 3528 and 3576 also had the 722 bp-fragment amplified (data not shown). 3.7. Spectrum of action of the bacteriocins produced by the CNS strains Strains 3154, 3528, 3576 and 3577 exhibited a spectrum of activity wider than the other strains (Table 4). Interestingly, the spectrum exhibited by
these strains was very similar to that exhibited by S. aureus A70, inhibiting all Listeria strains tested, an important food-borne and bovine mastitis associated pathogen, and Micrococcus. S. agalactiae, another common pathogen involved in bovine mastitis, was inhibited by strains 3154, 3528, 3576 and 3577. Strains 2166, 2167 and 3419 inhibited only C. fimi. 3.8. Cross-immunity against bacteriocins produced by S. aureus and S. epidermidis Protection of the bacteriocin-producer strains against their own bacteriocins is mediated by the so-called immunity peptides whose genetic determinants are generally found in the bacteriocin gene clusters. Immunity is generally bacteriocin specific. Strains that produce either identical or similar bacteriocins exhibit cross-immunity. Therefore, the presence of cross-immunity between bacteriocin producers is generally indicative of relatedness between their bacteriocins. The results of the cross-immunity tests performed in this study are shown in Table 5. Strains 3154, 3528 and 3576 were able to inhibit the growth of strain A70 Bac, cured of the plasmid pRJ6, but not of the wildtype strain; strain A70 did not inhibit strains 3154,
Fig. 1. PCR amplification of the aurABCD operon which encodes aureocin A70. Lanes: (A) DNA size marker; (B) S. aureus A70 (positive control); (C) 494; (D) 2166; (E) 2167; (F) 2457; (G) 3024; (H) 3154; (I) 3299; (J) 3414; (K) 3419; (L) 3528; (M) 3576; (N) 3577.
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Table 4 Spectrum of activity of the bacteriocins produced by coagulase-negative Staphylococcus strains Indicator strains
Producer strains
Bacillus megaterium F4 Corynebacterium fimi NCTC 7547 Enterococcus faecium E86 Lactobacillus casei Lactobacillus casei ATCC 393 Lactococcus lactis ATCC 11454 Leuconostoc lactis ATCC 19256 Listeria monocytogenes 11LM Listeria monocytogenes L1/2A Listeria monocytogenes 7848 Listeria innocua 397 Micrococcus spp. Paenibacillus polymyxa SCE-2 Pediococcus pentosaceus ATCC 43200 Streptococccus pyogenes Streptococcus agalactie
A70
494
2166
2167
2457
3024
3154
3299
3414
3419
3528
3576
3577
+ + NT t t t t + + + + + +
+ +
+
+
+ +
+ +
+ + + + + + +
+ + +
+ +
+
+ + + + + + +
+ + + + + + + +
+ + + + + + + +
(+) Inhibition; () no inhibition; NT, not tested; t, turbid zone of inhibition. Only the indicator strains that were inhibited by at least one of the producer strains are shown in this table. Enterococcus faecalis FA2-2, E. faecalis OG1X, Lactobacillus brevis ATCC 14869 and Leuconostoc mesenteroides ATCC 8293 were also tested, however, they were not inhibited by any of the producer strains.
3528 and 3576 suggesting the presence of crossimmunity among the bacteriocins produced by these four strains. Strain 3577 was also able to inhibit strain A70 Bac but not A70. However, strain A70 inhibited strain 3577, suggesting that the bacteriocins produced
by both strains are different. Strain 3299 inhibited only strain A53, indicating that its bacteriocin is distinct from aureocin A53. The producer strains of Pep5 and epidermin were able to inhibit all the 12 CNS strains associated with bovine mastitis, suggesting that none
Table 5 Immunity/resistance to bacteriocins among bacteriocinogenic CNS and S. aureus strains Indicator strains
S. S. S. S. S. S. S.
aureus A70 aureus A70 aureus A53 epidermidis epidermidis epidermidis epidermidis
Bac Tu¨ 3298 5 BN280 K7
Producer strains 494
2166
2167
2457
3024
3154
3299
3414
3419
3528
3576
3577
+ +
+
+ +
+ +
+
Producer strains
Indicator strains 494
2166
2167
2457
3024
3154
3299
3414
3419
3528
3576
3577
S. S. S. S. S. S.
+ +
+ +
+ + +
+ + + +
+ +
+ + +
+ + +
+ +
+ +
+ + +
+ + +
+ + +
aureus A70 aureus A53 epidermidis epidermidis epidermidis epidermidis
(aureocin A70) (aureocin A53) Tu¨ 3298 (epidermin) 5 (Pep5) BN280 (epicidin 280) K7 (epilancin K7)
(+) Inhibition; () no inhibition.
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Fig. 2. Inhibition of Streptococcus agalactiae strains involved in bovine mastitis by Bac+ coagulase-negative Staphylococcus strains. Seventyfour S. agalactiae strains involved in bovine mastitis were tested as indicators. (&) Number of inhibited strains and (&) percentage of inhibited strains.
SCN bacteriocin is related to Pep5 and epidermin. The producers of epicidin 280 and epilancin K7, however, were neither able to inhibit nor to be inhibited by any of the CNS strains. In such cases, the absence of inhibition could result from either bacteriocin immunity or resistance (i.e, inability of the bacteriocin to act on the cells). 3.9. Amplification of the structural genes for aureocin A53, epilancin K7 and epicidin 280 To discard the possibility that the CNS bacteriocins are related to aureocin A53, epilancin K7 and epicidin 280, PCR experiments for the amplification of the structural genes of these staphylococcins were performed and none of the 12 CNS strains involved in bovine mastitis showed amplification of the expected fragments (data not shown). 3.10. Inhibition of Streptococcus agalactiae strains involved in bovine mastitis Most of the S. agalactiae strains used as indicators (78.4%) were inhibited by strain 3299 (Fig. 2). Strains 2166, 2167, 3154, 3419 and 3577 were able to inhibit at least 40% of the indicators strains. The percentage of inhibition observed with the remaining strains lied between 10 and 38%.
4. Discussion Previous studies have shown the production of AMS by S. aureus strains from different sources.
Giambiagi-deMarval et al. (1990) found 13 (9.5%) AMS producers amongst 137 strains isolated from foods and not hospitalised patients. From 1990 to 1992, 163 S. aureus strains were isolated from different clinical specimens of human origin, in four hospitals from Rio de Janeiro. Fifty-three strains (32.5%) were shown to produce AMS, but only three (1.8%) presented a high antagonistic activity against C. fimi (Gamon et al., 1999). Oliveira et al. (1998a), studying 46 S. aureus strains isolated from apparently healthy cattle, identified only four (8.7%) bacteriocinproducer strains. Recently, Nascimento et al. (2002) studied 50 strains of S. aureus isolated from mastitic cows and verified that 24% of these strains showed a high inhibitory activity against C. fimi. Therefore, a higher incidence of AMS+ S. aureus strains was found amongst the isolates involved in bovine mastitis. In the present work, however, amongst 188 CNS, we found only 12 (6.4%) producer strains. AMS produced by most strains were sensitive to proteolytic enzymes suggesting they might be bacteriocins. The only exception was strain 3419. However, bacteriocins resistant to proteolytic digestion has already been reported in the literature (Netz et al., 2002). Although bacteriocin production is not a characteristic found in most Staphylococcus strains, it may confer a competitive advantage to the producer strains for nutrition in the surroundings, helping in the occupation of determined ecological niches. In this study, most of the Bac+ CNS strains were identified as S. epidermidis. The remaining strains were S. simulans, S. saprophyticus, S. hominis and S. arlettae. Bacteriocin production has already been described in S. epidermidis (Sahl and Bierbaum,
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1998). However, to our knowledge, this is the first report, which describes bacteriocin production in these four latter staphylococcal species. The resistance profile of the 12 CNS strains to 14 different antibiotics was also evaluated. In general, the strains did not present a great number of resistance phenotypes. These results contrast with those described for clinical isolates of CNS which generally carry multiple drug-resistance determinants (Aires de Sousa et al., 1998; Kohner et al., 1999; Santos et al., 1999). Only strain 3024 was shown to be resistant to six of the antibiotics tested, mainly b-lactamic drugs. The mecA gene, detected in this strain by PCR, is probably involved in resistance to the latter drugs. In Gram-positive bacteria, the genetic determinants involved in the Bac production have been found either on plasmids or on the bacterial chromosome (Jack et al., 1995). Amongst the 12 Bac+ CNS strains associated with bovine mastitis studied in this work, all strains presented at least one plasmid form. Except for S. hominis 3419, the genetic determinants encoding the bacteriocins could be either on the chromosome or on plasmids carried by them. In strain 3419, the bacteriocin genetic determinant is probably chromosomally encoded since the only plasmid found in this strain is too small to carry all genes generally required for bacteriocin expression (Ennahar et al., 2000; McAuliffe et al., 2001). Three strains (3154, 3528, 3576) were shown to possess a plasmid with a size similar to that of pRJ6 (8.0 kb), an S. aureus plasmid which encodes aureocin A70. PCR experiments were then carried out to test if the CNS strains presented the structural genes involved in aureocin A70 production. The amplification of the expected fragment of 525 bp was observed only in strains 3154, 3528 and 3576. These results indicate that the DNA of these strains possesses similar sequences to that found in the operon aurABCD. However, despite the amplification of the 525 bp fragment, it is not possible to assure that the amplified sequence is identical to that of pRJ6. This could be confirmed by DGGE (denaturing gradient gel electrophoresis) experiments, whose technique allows the detection of differences of even a single base-pair in DNA fragments with the same size (Muyzer et al., 1998). These experiments are currently in progress. To confirm that the 8.0 kb plasmids found in strains 3154, 3528 and 3576 are involved in bacteriocin
69
production, experiments of plasmid cure or transfer are required. However, hybridisation experiments with the HindIII-A fragment of pRJ6, the amplification of the bacteriocin operon as well as the amplification of the genes orfA and orfB, probably involved in the immunity to aureocin A70, are strong evidences that these plasmids are responsible for bacteriocin production, being either related or identical to pRJ6. For this reason, a more detailed characterisation of these plasmids was not performed. Previous studies (Giambiagi-deMarval et al., 1990; Oliveira et al., 1998a; Gamon et al., 1999; Nascimento et al., 2002) have shown that bacteriocinogenic plasmids of 8.0 kb are spread among the S. aureus population. However, this is the first report on the presence of this plasmid also among coagulase-negative staphylococci. Such results suggest that there is a certain level of transfer of plasmids related to pRJ6 among different species of this genus. Taking together these results support the conclusion that the bacteriocins produced by the CNS strains 3154, 3528 and 3576 are either identical or similar to aureocin A70. In relation to the remaining strains, the immunity/ resistance and PCR experiments showed that they produce bacteriocins which seem to be different from the most studied staphylococcins. However, experiments aiming the characterisation of these bacteriocins were not performed yet. The bacteriocins produced by the CNS strains presented a narrow spectrum of activity against Grampositive bacteria when compared to the bacteriocins produced by the S. aureus strains previously studied by our group (Giambiagi-deMarval et al., 1990; Oliveira et al., 1998a; Gamon et al., 1999). Only four strains (3154, 3528, 3576 and 3577) were able to inhibit L. monocytogenes, an important food-borne pathogen. However, when the ability of these strains to inhibit S. agalactiae strains involved in bovine mastitis was investigated, five CNS strains inhibited at least 50% of the indicator strains. These results suggest that the bacteriocins produced by some of these CNS strains, especially by S. simulans 3299, may be developed into useful antimicrobial drug for either treatment or prevention of bovine mastitis caused by Streptococcus, the second more important pathogen associated to this disease. However, applied studies must be done to confirm their effectiveness ‘‘in vivo’’. These studies must be preceded by bacteriocin
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purification and analysis of its biochemical structure. Since the bacteriocin produced by S. simulans 3299 seems to be the best candidate for prevention of streptococcal mastitis, the experiments aiming to its purification and characterisation are currently in progress.
Acknowledgements We thank Dr. Hans-Georg Sahl for sending us the S. epidermidis strains, which produce the bacteriocins Pep5, epidermin, epilancin K7 and epicidin 280. This work was supported by grants from CNPq, FAPERJ and PRONEX to M.C.F.B.
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heterologous expression of the novel lantibiotic epicidin 280 and analysis of its biosynthetic gene cluster. Appl. Environ. Microbiol. 64, 3140–3146. Jack, R.W., Tagg, J.R., Ray, B., 1995. Bacteriocins of Gram-positive bacteria. Microbiol. Rev. 59, 171–200. van de Kamp, M., van den Hooven, H.W., Konings, R.N., Bierbaum, G.A., Sahl, H.-G., Kuipers, O.P., Siezen, R.J., de Vos, W.M., Hilbers, C.W., van de Ven, F.J., 1995. Elucidation of the primary structure of the lantibiotic epilancin K7 from Staphylococccus epidermidis K7 Cloning and characterisation of the epilancinK7-encoding gene and NMR analysis of mature epilancin K7. Eur. J. Biochem. 230, 587–600. Kaletta, C., Entian, K.-D., Kellner, R., Jung, G., Reis, M., Sahl, H.G., 1989. Pep5, a new lantibiotic: structural gene isolation and prepeptide sequence. Arch. Microbiol. 152, 16–19. Kloos, W.E., Schleifer, K.H., 1986. Genus Staphylococcus. In: Sneath, P.H.A., Mair, N.S., Sharpe, M.E., Holt, J.G. (Eds.), Bergey’s Manual of Systematic Bacteriology, vol. 2. Williams & Wilkins, Baltimore, MD, pp. 1013–1035. Kohner, P., Uhl, J., Kolbert, C., Persing, D., Cockerill, F., 1999. Comparison of susceptibility testing methods with mecA gene analysis for determining oxacillin (methicillin) resistance in clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus spp. J. Clin. Microbiol. 37, 2952–2961. Lammers, A., van Vorstenbosch, C.J., Erkens, H.F., Smith, H.E., 2001. The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells. Vet. Microbiol. 80, 255–265. Leitner, G., Lubashevsky, E., Trainin, Z., 2003. Staphylococcus aureus vaccine against mastitis in dairy cows, composition and evaluation of its immunogenicity in a mouse model. Vet. Immunol. Immunopathol. 93, 159–167. McAuliffe, O., Ross, R.P., Hill, C., 2001. Lantibiotics: structure, biosynthesis and mode of action. FEMS Microbiol. Rev. 25, 285–308. Muyzer, G., Brinkhoff, T., Nu¨ bel, U., Santegoeds, C., Scha¨ fer, H., Wawer, C., 1998. Denaturing gradient gel electrophoresis (DGGE) in microbial ecology. Mol. Microbiol. Ecol. 3, 1–27. Nascimento, J.S., Santos, K.R.N., Gentilini, E., Sordelli, D., Bastos, M.C.F., 2002. Phenotypic and genetic characterisation of bacteriocin-producing strains of Staphylococcus aureus involved in bovine mastitis. Vet. Microbiol. 95, 133–144. National Committee for Clinical Laboratory Standards (NCCLS), 2002. Performance Standards for Antimicrobial Sensitivity Testing. NCCLS, Wayne, PA. Netz, D.J.A., Sahl, H.-G., Marcolino, R., Nascimento, J.S., Oliveira, S.S., Soares, M.B., Bastos, M.C.F., 2001. Molecular characterisation of aureocin A70, a multi-peptide bacteriocin from Staphylococcus aureus. J. Mol. Biol. 311, 939–949. Netz, D.J.A., Pohl, R., Beck-Sickinger, A.G., Selmer, T., Pierik, A.J., Bastos, M.C.F., Sahl, H.-G., 2002. Biochemical characterisation and genetic analysis of aureocin A53, a new, atypical bacteriocin from Staphylococcus aureus. J. Mol. Biol. 319, 745– 756. Nunes, E.L.C., Santos, K.R.N., Mondino, P.J.J., Bastos, M.C.F., Giambiagi-deMarval, M., 1999. Detection of ileS-2 gene encoding mupirocin resistance in methicillin-resistant Staphylococcus
J.S. Nascimento et al. / Veterinary Microbiology 106 (2005) 61–71 aureus by multiplex PCR. Diagn. Microbiol. Infect. Dis. 34, 77– 81. Oliveira, S.S., Po´ voa, D.C., Nascimento, J.S., Pereira, M.S.V., Siqueira Jr., J.P., Bastos, M.C.F., 1998a. Antimicrobial substances produced by Staphylococcus aureus strains isolated from cattle in Brazil. Lett. Appl. Microbiol. 27, 229–234. Oliveira, S.S., Abrantes, J., Cardoso, M., Sordelli, D., Bastos, M.C.F., 1998b. Staphylococcal strains involved in bovine mastitis are inhibited by Staphylococcus aureus antimicrobial peptides. Lett. Appl. Microbiol. 27, 287–291. Riffon, R., Sayasith, K., Khalil, H., Dubreuil, P., Drolet, M., Lagace´ , J., 2001. Development of a rapid and sensitive test for identification of major pathogens in bovine mastitis by PCR. J. Clin. Microbiol. 39, 2584–2589. Sahl, H.-G., Bierbaum, G., 1998. Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from Grampositive bacteria. Annu. Rev. Microbiol. 52, 41–79. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.
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Santos, K.R.N., Teixeira, L.M., Leal, G.S., Fonseca, L.S., Gontijo Filho, P.P., 1999. DNA typing of methicillin-resistant Staphylococcus aureus: isolates and factors associated with nosocomial acquisition in two Brazilian university hospitals. J. Med. Microbiol. 48, 17–23. Soltys, J., Quinn, M.T., 1999. Selective recruitment of T-cell subsets to the udder during staphylococcal and streptococcal mastitis: analysis of lymphocyte subsets and adhesion molecule expression. Infect. Immun. 67, 6293–6302. Sordelli, D.O., Buzzola, F.R., Gomez, M.I., Steele-Moore, L., Berg, D., Gentilini, E., Catalano, M., Reitz, A.J., Tollersrud, T., Denamiel, G., Jeric, P., Lee, J.C., 2000. Capsule expression by bovine isolates of Staphylococcus aureus from Argentina: genetic and epidemiologic analyses. J. Clin. Microbiol. 38, 846–850. Younis, A., Krifucks, O., Heller, E.D., Samra, Z., Glickman, A., Saran, A., Leitner, G., 2003. Staphylococcus aureus exosecretions and bovine mastitis. J. Vet. Med. 50, 1–7. Zhang, S., Maddox, C.W., 2000. Cytotoxic activity of coagulasenegative staphylococci in bovine mastitis. Infect. Immun. 68, 1102–1108.
Production of bacteriocins by coagulase-negative staphylococci involved in bovine mastitis Janaı´na dos Santos Nascimento a, Patricia Carlin Fagundes a, Maria Aparecida Vasconcelos de Paiva Brito b, Ka´tia Regina Netto dos Santos a, Maria do Carmo de Freire Bastos a,* a
Instituto de Microbiologia Prof. Paulo de Go´es, Departamento de Microbiologia Geral, Universidade do Brazil (UFRJ), CCS, Bloco I, sala I-1-059, Cidade Universita´ria, 21941-590 Rio de Janeiro, RJ, Brazil b EMBRAPA Gado de Leite, Juiz de Fora, MG, Brasil Received 8 June 2004; received in revised form 20 September 2004; accepted 10 October 2004
Abstract In the present study, 188 coagulase-negative Staphylococcus (CNS) strains were isolated from bovine mastitis cases from 56 different Brazilian dairy herds, located in the Southeast region of the country, and were tested for antimicrobial substance production. Twelve CNS strains (6.4%) exhibited antagonistic activity against a Corynebacterium fimi indicator strain. Most antimicrobial substances were sensitive to proteolytic enzymes suggesting that they might be bacteriocins (Bac). Amongst the CNS producers, six were identified as S. epidermidis, two as S. simulans, two as S. saprophyticus, one as S. hominis and one as S. arlettae. Plasmid profile analysis of these strains revealed the presence of at least one plasmid. The Bac+ strains presented either no or few antibiotic resistance phenotypes. Three strains were shown to produce a bacteriocin either identical or similar to aureocin A70, a bacteriocin previously isolated from an S. aureus strain isolated from food. The remaining Bac+ strains produce antimicrobial peptides that seem to be distinct from the best characterised staphylococcal bacteriocins described so far. Some of them were able to inhibit Listeria monocytogenes, an important food-borne pathogen, and several strains of Streptococcus agalactiae associated with bovine mastitis, suggesting a potential use of these bacteriocins either in the prevention or in the treatment of streptococcal mastitis. # 2004 Elsevier B.V. All rights reserved. Keywords: Coagulase-negative Staphylococcus spp.; Bacteriocin; Bovine mastitis; Cattle-bacteria
1. Introduction Bovine mastitis is an inflammation of the mammary glands usually due to a microbial infection * Corresponding author. Fax: +55 21 2560 8344. E-mail address: [email protected] (M.C.F. Bastos).
that affects milk production and quality, being one of the most significant causes of economic loss to the dairy industry (Soltys and Quinn, 1999; Sordelli et al., 2000; Riffon et al., 2001; Bradley, 2002). Although several bacterial pathogens can cause mastitis, Staphylococcus aureus is the most important causative agent (followed by Streptococcus spp.), and
0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2004.10.014
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once it is established in the mammary glands of the animal, it is very difficult to eradicate (Soltys and Quinn, 1999; Lammers et al., 2001). Coagulase-negative staphylococci (CNS) have been considered a minor pathogen of bovine mastitis. However, many studies have recently shown the importance of CNS infection of the bovine mammary glands (Soltys and Quinn, 1999; Zhang and Maddox, 2000; Younis et al., 2003). In the efforts to avoid mastitis, some vaccines that can reduce the severity of this illness were generated. However, these vaccines still do not control efficiently the development of mastitis (Leitner et al., 2003). On the other hand, it has been verified that the indiscriminate treatment with antibiotics, without either a technical prescription or identification tests of the pathogen, can contribute to an increased resistance of these micro-organisms, making the cure of mastitis still more difficult (Gruet et al., 2001). Moreover, it is important to stand out that these medicines are set free in milk for some days, after their administration, being able to cause problems for consumers. Thus, the identification of alternative methods for controlling this illness is essential. One of these methods could be the use of bacteriocins. Bacteriocins (Bac) are antimicrobial peptides or proteins produced by bacteria with inhibitory activity against other bacteria. These substances, specially those produced by Gram-positive bacteria, present a potential use either in food industries as biopreservatives or in the prevention and treatment of some infectious diseases, having medical and veterinary applications (Jack et al., 1995; Oliveira et al., 1998b; Sahl and Bierbaum, 1998). Amongst the bacteriocin-producer staphylococcal species, S. epidermidis is the most studied. Some of its bacteriocins are very well characterised, such as Pep5, epidermin, epicidin 280 and epilancin K7, which are classified as lantibiotics. Pep5 is a 34-amino acid peptide encoded by the structural gene pepA (Kaletta et al., 1989). Epidermin is also a well-characterised lantibiotic produced by S. epidermidis Tu¨ 3298. Its structural gene, epiA, codes for a 22-amino acid peptide (Augustin et al., 1992). Epicidin 280 is also a 30-amino acid lantibiotic, produced by S. epidermidis BN280, which has 75% of homology with Pep5 (Heidrich et al., 1998). Epilancin K7, a 31-amino acid lantibiotic produced by S. epidermidis K7, is encoded by the gene elkA (van de Kamp et al., 1995).
Our laboratory has been investigating bacteriocin production by Staphylococcus strains, which is generally associated to plasmids. Amongst the bacteriocins produced by S. aureus and studied by our group, aureocins A70 and A53 are the best characterised. Aureocin A70 is a multi-peptide, nonlantibiotic bacteriocin produced by S. aureus A70 which is encoded by an 8.0 kb-plasmid named pRJ6 (Giambiagi-deMarval et al., 1990). Aureocin A70 was the first bacteriocin described which is composed of four related small cationic peptides, which are encoded by the aurABCD operon (Netz et al., 2001). Aureocin A53 is a 51-amino acid peptide produced by S. aureus A53. It is encoded on a 10.4 kb plasmid, pRJ9 (Netz et al., 2002). As well as other antimicrobial substances, bacteriocins produced by staphylococci (staphylococcins) could be employed, in their purified form, on the control of bovine mastitis. Therefore, in the present study, in an attempt to detect new bacteriocins with potential medical and veterinary applications, we extended our studies on bacteriocin production to CNS strains, which were associated with bovine mastitis in different Brazilian dairy herds.
2. Materials and methods 2.1. Bacterial strains and culture conditions One hundred and eighty-eight coagulase-negative Staphylococcus strains involved in bovine mastitis were isolated from 56 different Brazilian dairy herds located in the Southeast region of the country, and used in the screening for bacteriocin production. Seventy-four Streptococcus agalactiae strains were also isolated from bovine mastitis cases and used as indicators. These strains were isolated according to procedures recommended by Harmon et al. (1990). S. aureus and S. epidermidis strains from previous studies (Table 1) were used either as producers or as indicators in the inhibition assays. Staphylococcus strains were grown in either TSB (Difco) or BHI (Difco), at 37 8C for 18 h. TSB was used to grow the strains for DNA isolation and BHI was used in all bacteriocin assays. The bacteria used as indicators were grown in BHI medium, except for lactic acid bacteria, that were cultivated in MRS medium (BBL)
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Table 1 Staphylococcus strains previously described and used in this study Strain
Relevant features
Reference/source
Staphylococcus aureus A53 A70 A70 Bac
Bac+ (aureocin A53), Imm+, plasmid: pRJ9 (10.4 kb) Bac+ (aureocin A70), Imm+, plasmid: pRJ6 (8.0 kb) Strain A70 cured of pRJ6
Giambiagi-deMarval et al. (1990) Giambiagi-deMarval et al. (1990) Giambiagi-deMarval et al. (1990)
Staphylococcus epidermidis Tu¨ 3298 5 5 (cured) BN280 K7
Bac+ (epidermin) Bac+ (Pep5) Bac Pep5 Bac+ (epicidin 280) Bac+ (epilancin K7)
Augustin et al. (1992) Kaletta et al. (1989) Bierbaum et al. (1994) Heidrich et al. (1998) van de Kamp et al. (1995)
Bac, bacteriocin; Imm, immunity.
and incubated at 28 8C for 48 h under microaerophilic atmosphere. Bacteria were stored in their appropriate culture medium with 40% glycerol (w/v) at 20 8C until needed. When necessary, the media were supplemented with agar at 1.5% (w/v) or 0.6% (w/v).
(1990). Lack of inhibition zones when C. fimi NCTC7547 was used as the indicator strain indicated that the antimicrobial compound had either a proteinaceous or an acidic nature. 2.5. Antibiotic resistance
2.2. Assay for antimicrobial substance production This assay was done as described previously by Giambiagi-deMarval et al. (1990). C. fimi NCTC 7547 was used as the indicator strain for production of antimicrobial substances. 2.3. Identification of the strains Only the CNS strains that exhibited antimicrobial substance production were identified, using conventional biochemical tests (Kloos and Schleifer, 1986) or, when necessary, using a commercial kit for identification (API Staph, BioMe´ rieux). S. agalactiae strains were identified on the basis of Gram staining, presence of hemolysis, negative results on tests for the presence of catalase and esculin hydrolysis, and positive results on tests for CAMP and hydrolysis of sodium hypurate. 2.4. Effects of proteolytic enzymes and of 0.2N NaOH on AMS activity
Resistance patterns were determined by disc diffusion on Mueller-Hinton agar (Oxoid), according to the National Committee for Clinical Laboratory Standards (NCCLS, 2002). The following antibiotics (Sensifar) were used: ampicillin (10 mg), cephalothin (30 mg), ciprofloxacin (5 mg), clindamycin (2 mg), chloramphenicol (30 mg), erythromycin (15 mg), gentamicin (10 mg), imipenen (10 mg), mupirocin (5 mg), oxacillin (1 mg), penicillin (10 U), rifampin (5 mg), tetracycline (30 mg) and vancomycin (30 mg). The diameters of the inhibition zones were interpreted according to the NCCLS guidelines after 24 h of incubation at 37 8C. Strains which showed resistance to b-lactamic drugs were also tested for the presence of the methicillin-resistance gene (mecA) by PCR. 2.6. Isolation of plasmid DNA Whole-cell lysates were prepared as described by Giambiagi-deMarval et al. (1990). 2.7. DNA–DNA hybridisation assays
The effects of trypsin (Sigma), proteinase K (Boehinger Mannheim), protease XXIII (Sigma) and 0.2N NaOH on AMS activity were determined by the method described by Giambiagi-deMarval et al.
Southern blots and hybridisations were performed as described by Bastos and Murphy (1988). The 5.2-kb HindIII-A fragment of pRJ6 (encompassing all genes
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required for aureocin A70 production) was used as a probe. 32P-labelled probes were prepared by the Random-Primers DNA Labelling System (Invitrogen) following the manufacturer’s recommendations.
inhibitory action of the AMS produced by the CNS strains against S. agalactiae involved in bovine mastitis was also tested using 74 indicator strains.
2.8. PCR amplification
2.10. Agarose gel electrophoresis
Total DNA was extracted by boiling, as described previously by Nunes et al. (1999). The primers used for amplification of the aurABCD operon and the genes aucA, eciA and elkA (the structural genes of aureocin A70, aureocin A53, epicidin 280 and epilancin K7, respectively) as well as for amplification of the methicillin-resistance gene (mecA) and genes orfA and orfB (presumably involved in the immunity to aureocin A70) are listed in Table 2. Each PCR reaction contained: 1 PCR reaction buffer (Invitrogen), 2.5 mM concentration of each deoxyribonucleoside triphosphate, 2.5 U of Taq-Polymerase (Invitrogen) and 50 pmol of each primer. The amplification was done in a Programmable Thermal Controller (PTC-100TM, MJ Research, USA) and the cycles were performed as described by Nunes et al. (1999). A 100bp DNA ladder (Invitrogen) was used as the molecular size marker.
Agarose gel electrophoreses were performed as described by Sambrook et al. (1989). Products amplified by PCR were analysed in 1.4% (w/v) agarose gels and the plasmid profiles were determined in 0.7% (w/v) agarose gels. The gels were stained in an aqueous solution containing ethidium bromide (0.5 mg/ml) and visualised on a UV transiluminator.
2.9. Determination of the inhibitory spectrum of each AMS+ strain To determine the inhibitory spectrum of each AMS, several strains from different species of Grampositive bacteria were tested as indicators. The
3. Results 3.1. Detection and identification of the AMS+ strains One hundred and eighty-eight coagulase-negative Staphylococcus (CNS) strains were isolated from bovine mastitis cases from 56 different Brazilian dairy herds, located in the Southeast region of the country, and were tested for AMS production. Twelve CNS strains (6.4%), belonging to six different herds, exhibited antimicrobial substance production against C. fimi (inhibition zones between 17 and 38 mm). Among the CNS producers, six were identified as S. epidermidis, two as S. simulans, two as S. saprophyticus, one as S. hominis and one as S. arlettae (Table 3).
Table 2 Primers used in this work Gene(s) to be amplified
Relevant features
Primers
Size (bp) of the fragment to be amplified
aurABCD
Aureocin A70 structural genes
525
aucA
Aureocin A53 structural gene
orfA and orfB
Immunity genes to aureocin A70
elkA
Epilancin K7 structural gene
eciA
Epicidin 280 structural gene
mecA
Methicillin-resistance gene
Forward: P4B, 50 -CCTTATAACTTCGAATGCT-30 ; reverse: P5, 50 -AATTATTAACAAGAGAAA-30 Forward: AUC1, 50 -GAAGTTGTGAAAACTATTA-30 ; reverse: AUC2, 50 -CATAAAACAAAGAGCCAAAGT-30 Forward: RJ61, 50 -GACGAGGGTATTGCATA-30 ; reverse: RJ62, 50 -CTAAGCATTCGATAAGG-30 Forward: K71, 50 -ATGAATAACTCATTATTC-30 ; reverse: K72, 50 -ATGGAA AACAAAAAAG-30 Forward: Epin1, 50 -CAGGAG GGATATATTATGG-30 ; reverse: Epin2, 50 -CAATCACTACTATTGACAATCAC-30 Forward: MRS1, 50 -TAGAAATGA CTG AACGTCCG-30 ; reverse: MRS2, 50 -TTGCGATCAATGTTACCG TAG-30
322 722 171 165 154
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Table 3 Characteristics of the bacteriocin-producer CNS strains Strains b
A70 494 2166 2167 2457 3024 3154 3299 3414 3419 3528 3576 3577
Identification
S. S. S. S. S. S. S. S. S. S. S. S. S.
aureus epidermidis epidermidis epidermidis simulans epidermidis saprophyticus simulans epidermidis hominis arlettae saprophyticus epidermidis
Sensitivity of the Bac to proteolytic enzymes Protease XXIII
Proteinase K
Trypsin
S R R S S S R R R R S S S
S S S R R S S S S R S S R
S S S R S S S S S R S S R
Resistance profile
Plasmid formsa
Emc Emc Emc Ap, Ce, Gm, Ox, Pc, Tc Emc Ap, Pc Ap, Pc
8.0 (pRJ6) >25; 15; 6.7; 5.5; 3.7; 3.2; 2.2 12; 6.2 12; 6.2 >27 15; 8.1; 4.5 15; 8 >25; 3.5 >25 4 15; 8 15; 8 >25 (2)d; 2.5; 1.5
R, resistant; S, sensitive; () sensitive to all the antibiotics tested; Ap, ampicillin; Ce, cephalothin; Em, erythromycin; Gm, gentamicin; Ox, oxacillin; Pc, penicillin; Tc, tetracycline. a Size in kb. b Strain A70 was included in these experiments as a positive control. c Intermediate resistance. d The number between parentheses indicates the presence of two plasmid bands with size larger than 25 kb.
3.2. Effects of proteolytic enzymes on AMS activity The results are also presented in Table 3. The AMS were resistant to 0.2N NaOH, discarding the possibility that the inhibition exhibited was due to organic acids produced by the producer strain during its metabolism. Except for the AMS produced by the strain 3419, the remaining AMS were sensitive to at least one proteolytic enzyme tested, indicating that these AMS present a biological active proteinaceus component in their structure, the main characteristic of a typical bacteriocin. Therefore, from hereafter, the AMS+ strains will be considered Bac+. Bacteriocins produced by strains 3154, 3528 and 3576, as well as aureocin A70, showed a high sensitivity to proteolytic enzymes, being sensitive to all enzymes tested. 3.3. Antibiotic resistance profile The Bac+ strains did not exhibit a great number of resistance markers, except for strain 3024, that showed resistance to six antibiotics (Table 3). The remaining strains were resistant to either two (ampicillin and penicillin) or none of the antibiotics tested. Strains 494, 2167, 2457 and 3154 presented intermediate resistance to erythromycin. Strains that showed
resistance to one or more b-lactamic drugs were also tested for the presence of the mecA gene, which encodes methicillin resistance. Among the strains tested, only strain 3024 showed amplification of the mecA gene (data not shown). 3.4. Plasmid profiles All strains showed at least one plasmid form (Table 3). Strains 3154 (S. saprophyticus), 3528 (S. arlettae) and 3576 (S. saprophyticus) exhibited an identical plasmid profile, and, interestingly, they carry a plasmid with a size similar to that of pRJ6 (8.0 kb), a bacteriocinogenic plasmid previously characterised by our group and that encodes aureocin A70. These three strains were isolated from the same herd. Strains 2166 and 2167 (both S. epidermidis and isolated from the same herd), also presented an identical plasmid profile. 3.5. DNA–DNA hybridisation assays Since strains 3154, 3528 and 3576 carry a plasmid with a size similar to that of pRJ6, the homology between these plasmids and pRJ6 was investigated. A strong signal was detected to the 8.0-kb plasmids
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carried by strains 3154, 3528 and 3576. No hybridisation was observed in the remaining CNS strains. 3.6. Amplification of the aurABCD and orfAB operons The presence or the absence of the aureocin A70 structural genes was also confirmed by PCR using specific primers from sequences flanking operon aurABCD. The host strain of pRJ6, S. aureus A70, was used as a positive control. Strains 3154, 3528 and 3576 had a 525-bp fragment amplified. The remaining nine strains did not present amplification of this fragment (Fig. 1). Additionally, PCR experiments using primers to amplify the genes orfA and orfB, that seem to be involved in the immunity to aureocin A70, revealed that only strains 3154, 3528 and 3576 also had the 722 bp-fragment amplified (data not shown). 3.7. Spectrum of action of the bacteriocins produced by the CNS strains Strains 3154, 3528, 3576 and 3577 exhibited a spectrum of activity wider than the other strains (Table 4). Interestingly, the spectrum exhibited by
these strains was very similar to that exhibited by S. aureus A70, inhibiting all Listeria strains tested, an important food-borne and bovine mastitis associated pathogen, and Micrococcus. S. agalactiae, another common pathogen involved in bovine mastitis, was inhibited by strains 3154, 3528, 3576 and 3577. Strains 2166, 2167 and 3419 inhibited only C. fimi. 3.8. Cross-immunity against bacteriocins produced by S. aureus and S. epidermidis Protection of the bacteriocin-producer strains against their own bacteriocins is mediated by the so-called immunity peptides whose genetic determinants are generally found in the bacteriocin gene clusters. Immunity is generally bacteriocin specific. Strains that produce either identical or similar bacteriocins exhibit cross-immunity. Therefore, the presence of cross-immunity between bacteriocin producers is generally indicative of relatedness between their bacteriocins. The results of the cross-immunity tests performed in this study are shown in Table 5. Strains 3154, 3528 and 3576 were able to inhibit the growth of strain A70 Bac, cured of the plasmid pRJ6, but not of the wildtype strain; strain A70 did not inhibit strains 3154,
Fig. 1. PCR amplification of the aurABCD operon which encodes aureocin A70. Lanes: (A) DNA size marker; (B) S. aureus A70 (positive control); (C) 494; (D) 2166; (E) 2167; (F) 2457; (G) 3024; (H) 3154; (I) 3299; (J) 3414; (K) 3419; (L) 3528; (M) 3576; (N) 3577.
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Table 4 Spectrum of activity of the bacteriocins produced by coagulase-negative Staphylococcus strains Indicator strains
Producer strains
Bacillus megaterium F4 Corynebacterium fimi NCTC 7547 Enterococcus faecium E86 Lactobacillus casei Lactobacillus casei ATCC 393 Lactococcus lactis ATCC 11454 Leuconostoc lactis ATCC 19256 Listeria monocytogenes 11LM Listeria monocytogenes L1/2A Listeria monocytogenes 7848 Listeria innocua 397 Micrococcus spp. Paenibacillus polymyxa SCE-2 Pediococcus pentosaceus ATCC 43200 Streptococccus pyogenes Streptococcus agalactie
A70
494
2166
2167
2457
3024
3154
3299
3414
3419
3528
3576
3577
+ + NT t t t t + + + + + +
+ +
+
+
+ +
+ +
+ + + + + + +
+ + +
+ +
+
+ + + + + + +
+ + + + + + + +
+ + + + + + + +
(+) Inhibition; () no inhibition; NT, not tested; t, turbid zone of inhibition. Only the indicator strains that were inhibited by at least one of the producer strains are shown in this table. Enterococcus faecalis FA2-2, E. faecalis OG1X, Lactobacillus brevis ATCC 14869 and Leuconostoc mesenteroides ATCC 8293 were also tested, however, they were not inhibited by any of the producer strains.
3528 and 3576 suggesting the presence of crossimmunity among the bacteriocins produced by these four strains. Strain 3577 was also able to inhibit strain A70 Bac but not A70. However, strain A70 inhibited strain 3577, suggesting that the bacteriocins produced
by both strains are different. Strain 3299 inhibited only strain A53, indicating that its bacteriocin is distinct from aureocin A53. The producer strains of Pep5 and epidermin were able to inhibit all the 12 CNS strains associated with bovine mastitis, suggesting that none
Table 5 Immunity/resistance to bacteriocins among bacteriocinogenic CNS and S. aureus strains Indicator strains
S. S. S. S. S. S. S.
aureus A70 aureus A70 aureus A53 epidermidis epidermidis epidermidis epidermidis
Bac Tu¨ 3298 5 BN280 K7
Producer strains 494
2166
2167
2457
3024
3154
3299
3414
3419
3528
3576
3577
+ +
+
+ +
+ +
+
Producer strains
Indicator strains 494
2166
2167
2457
3024
3154
3299
3414
3419
3528
3576
3577
S. S. S. S. S. S.
+ +
+ +
+ + +
+ + + +
+ +
+ + +
+ + +
+ +
+ +
+ + +
+ + +
+ + +
aureus A70 aureus A53 epidermidis epidermidis epidermidis epidermidis
(aureocin A70) (aureocin A53) Tu¨ 3298 (epidermin) 5 (Pep5) BN280 (epicidin 280) K7 (epilancin K7)
(+) Inhibition; () no inhibition.
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Fig. 2. Inhibition of Streptococcus agalactiae strains involved in bovine mastitis by Bac+ coagulase-negative Staphylococcus strains. Seventyfour S. agalactiae strains involved in bovine mastitis were tested as indicators. (&) Number of inhibited strains and (&) percentage of inhibited strains.
SCN bacteriocin is related to Pep5 and epidermin. The producers of epicidin 280 and epilancin K7, however, were neither able to inhibit nor to be inhibited by any of the CNS strains. In such cases, the absence of inhibition could result from either bacteriocin immunity or resistance (i.e, inability of the bacteriocin to act on the cells). 3.9. Amplification of the structural genes for aureocin A53, epilancin K7 and epicidin 280 To discard the possibility that the CNS bacteriocins are related to aureocin A53, epilancin K7 and epicidin 280, PCR experiments for the amplification of the structural genes of these staphylococcins were performed and none of the 12 CNS strains involved in bovine mastitis showed amplification of the expected fragments (data not shown). 3.10. Inhibition of Streptococcus agalactiae strains involved in bovine mastitis Most of the S. agalactiae strains used as indicators (78.4%) were inhibited by strain 3299 (Fig. 2). Strains 2166, 2167, 3154, 3419 and 3577 were able to inhibit at least 40% of the indicators strains. The percentage of inhibition observed with the remaining strains lied between 10 and 38%.
4. Discussion Previous studies have shown the production of AMS by S. aureus strains from different sources.
Giambiagi-deMarval et al. (1990) found 13 (9.5%) AMS producers amongst 137 strains isolated from foods and not hospitalised patients. From 1990 to 1992, 163 S. aureus strains were isolated from different clinical specimens of human origin, in four hospitals from Rio de Janeiro. Fifty-three strains (32.5%) were shown to produce AMS, but only three (1.8%) presented a high antagonistic activity against C. fimi (Gamon et al., 1999). Oliveira et al. (1998a), studying 46 S. aureus strains isolated from apparently healthy cattle, identified only four (8.7%) bacteriocinproducer strains. Recently, Nascimento et al. (2002) studied 50 strains of S. aureus isolated from mastitic cows and verified that 24% of these strains showed a high inhibitory activity against C. fimi. Therefore, a higher incidence of AMS+ S. aureus strains was found amongst the isolates involved in bovine mastitis. In the present work, however, amongst 188 CNS, we found only 12 (6.4%) producer strains. AMS produced by most strains were sensitive to proteolytic enzymes suggesting they might be bacteriocins. The only exception was strain 3419. However, bacteriocins resistant to proteolytic digestion has already been reported in the literature (Netz et al., 2002). Although bacteriocin production is not a characteristic found in most Staphylococcus strains, it may confer a competitive advantage to the producer strains for nutrition in the surroundings, helping in the occupation of determined ecological niches. In this study, most of the Bac+ CNS strains were identified as S. epidermidis. The remaining strains were S. simulans, S. saprophyticus, S. hominis and S. arlettae. Bacteriocin production has already been described in S. epidermidis (Sahl and Bierbaum,
J.S. Nascimento et al. / Veterinary Microbiology 106 (2005) 61–71
1998). However, to our knowledge, this is the first report, which describes bacteriocin production in these four latter staphylococcal species. The resistance profile of the 12 CNS strains to 14 different antibiotics was also evaluated. In general, the strains did not present a great number of resistance phenotypes. These results contrast with those described for clinical isolates of CNS which generally carry multiple drug-resistance determinants (Aires de Sousa et al., 1998; Kohner et al., 1999; Santos et al., 1999). Only strain 3024 was shown to be resistant to six of the antibiotics tested, mainly b-lactamic drugs. The mecA gene, detected in this strain by PCR, is probably involved in resistance to the latter drugs. In Gram-positive bacteria, the genetic determinants involved in the Bac production have been found either on plasmids or on the bacterial chromosome (Jack et al., 1995). Amongst the 12 Bac+ CNS strains associated with bovine mastitis studied in this work, all strains presented at least one plasmid form. Except for S. hominis 3419, the genetic determinants encoding the bacteriocins could be either on the chromosome or on plasmids carried by them. In strain 3419, the bacteriocin genetic determinant is probably chromosomally encoded since the only plasmid found in this strain is too small to carry all genes generally required for bacteriocin expression (Ennahar et al., 2000; McAuliffe et al., 2001). Three strains (3154, 3528, 3576) were shown to possess a plasmid with a size similar to that of pRJ6 (8.0 kb), an S. aureus plasmid which encodes aureocin A70. PCR experiments were then carried out to test if the CNS strains presented the structural genes involved in aureocin A70 production. The amplification of the expected fragment of 525 bp was observed only in strains 3154, 3528 and 3576. These results indicate that the DNA of these strains possesses similar sequences to that found in the operon aurABCD. However, despite the amplification of the 525 bp fragment, it is not possible to assure that the amplified sequence is identical to that of pRJ6. This could be confirmed by DGGE (denaturing gradient gel electrophoresis) experiments, whose technique allows the detection of differences of even a single base-pair in DNA fragments with the same size (Muyzer et al., 1998). These experiments are currently in progress. To confirm that the 8.0 kb plasmids found in strains 3154, 3528 and 3576 are involved in bacteriocin
69
production, experiments of plasmid cure or transfer are required. However, hybridisation experiments with the HindIII-A fragment of pRJ6, the amplification of the bacteriocin operon as well as the amplification of the genes orfA and orfB, probably involved in the immunity to aureocin A70, are strong evidences that these plasmids are responsible for bacteriocin production, being either related or identical to pRJ6. For this reason, a more detailed characterisation of these plasmids was not performed. Previous studies (Giambiagi-deMarval et al., 1990; Oliveira et al., 1998a; Gamon et al., 1999; Nascimento et al., 2002) have shown that bacteriocinogenic plasmids of 8.0 kb are spread among the S. aureus population. However, this is the first report on the presence of this plasmid also among coagulase-negative staphylococci. Such results suggest that there is a certain level of transfer of plasmids related to pRJ6 among different species of this genus. Taking together these results support the conclusion that the bacteriocins produced by the CNS strains 3154, 3528 and 3576 are either identical or similar to aureocin A70. In relation to the remaining strains, the immunity/ resistance and PCR experiments showed that they produce bacteriocins which seem to be different from the most studied staphylococcins. However, experiments aiming the characterisation of these bacteriocins were not performed yet. The bacteriocins produced by the CNS strains presented a narrow spectrum of activity against Grampositive bacteria when compared to the bacteriocins produced by the S. aureus strains previously studied by our group (Giambiagi-deMarval et al., 1990; Oliveira et al., 1998a; Gamon et al., 1999). Only four strains (3154, 3528, 3576 and 3577) were able to inhibit L. monocytogenes, an important food-borne pathogen. However, when the ability of these strains to inhibit S. agalactiae strains involved in bovine mastitis was investigated, five CNS strains inhibited at least 50% of the indicator strains. These results suggest that the bacteriocins produced by some of these CNS strains, especially by S. simulans 3299, may be developed into useful antimicrobial drug for either treatment or prevention of bovine mastitis caused by Streptococcus, the second more important pathogen associated to this disease. However, applied studies must be done to confirm their effectiveness ‘‘in vivo’’. These studies must be preceded by bacteriocin
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purification and analysis of its biochemical structure. Since the bacteriocin produced by S. simulans 3299 seems to be the best candidate for prevention of streptococcal mastitis, the experiments aiming to its purification and characterisation are currently in progress.
Acknowledgements We thank Dr. Hans-Georg Sahl for sending us the S. epidermidis strains, which produce the bacteriocins Pep5, epidermin, epilancin K7 and epicidin 280. This work was supported by grants from CNPq, FAPERJ and PRONEX to M.C.F.B.
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