Bacteriocin production by clinical Staphylococcus isolates

B. Robredo¹, I. Olarte² and C. Torres¹

¹Área de Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain;

²Sección de Microbiología, Hospital San Millán, Logroño, Spain

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria; they inhibit the growth of strains and species usually related to bacteriocin-producing bacteria. Although the majority of research has focused on bacteriocins produced by lactic acid bacteria (1) due to their potential for food preservation, there are several reports of the existence of bacteriocins in Staphylococcus aureus (2-5) and coagulase-negative staphylococci (6, 7). However, few reports describe bacteriocin production in Staphylococcus spp. of clinical origin (8, 9). The objective of this study was to detect bacteriocin production in methicillin-resistant and methicillin-susceptible staphylococci isolated from clinical samples, in order to determine their spectrum activity and to characterize their inhibitory activity.

One hundred and seven Staphylococcus clinical isolates collected in a 450-bed primary care hospital in Logroño, Spain, during April and May 2000, were tested for antimicrobial activity. The isolates were identified as follows: 99 S. aureus (41 methicillin-resistant and 58 methicillin-susceptible); seven Staphylococcus epidermidis (two methicillin-resistant and five methicillin-susceptible); and an atypical Staphylococcus strain which was coagulase-negative but DNAase positive (ST35). Fifty-eight bacterial isolates belonging to eight different genera and 23 different species were used as indicator strains for bacteriocin production (Table 1). Antimicrobial activity was evaluated using the method previously described by Navarro et al. (10) with some modifications. Plates containing Tripticase Soy Broth, plus 0.5% yeast extract and 1.5% agar (TSAYE, Difco) were spotted with the putative bacteriocin-producing strains and incubated overnight at 37 °C. Five-milliliter aliquots of brain heart infusion (Difco) containing 0.8% agar were maintained at 45 °C and seeded with 40 ml of the overnight cultures of the isolates used as indicators for bacteriocin detection.

The spotted TSAYE agar plates were carpeted with indicator strains and incubated at 37 °C for 24 h. Growth inhibition was detected by a zone of clearing around the producer strain.

Only two of the 107 studied isolates showed antimicrobial activity: S. aureus ST50 (methicillin-resistant and penicillin-resistant, isolated from a blood sample) and coagulase-negative Staphylococcus ST35 (methicillin-susceptible and penicillin-susceptible, isolated from a gastrointestinal sample from an immunocompromised patient). Results obtained by API Staph system (BioMérieux), as well as by other biochemical tests (Bergey's), identified the ST35 isolate as a probable S. epidermidis. Both strains were susceptible to all the other 16 tested antibiotics, including glycopeptides, aminoglycosides, tetracyclines, macrolides, lincosamides, rifampicin, novobiocin, quinolones, b-lactams, quinolones, trimethoprim, and sulfamethoxazole. Unlike coagulase-negative Staphylococcus ST35, S. aureus ST50 showed b-hemolysis when sheep blood was added to the growth media. Table 1 shows the spectrum of antimicrobial activity of both strains against the 58 isolates studied.

S. aureus ST50 inhibited the growth of nine out of 12 (75%) methicillin-resistant staphylococci (all S. aureus), and of two out of 11 (18%) of the methicillin-susceptible staphylococci tested. It is notewothy that a high percentage of methicillin-resistant Staphylococcus isolates were inhibited by S. aureus ST50. Curiously, S. aureus ST50 produced a bacteriocin active against Enterococcus durans, but no inhibitory growth activity against any of the other Enterococcus species or other genera studied was detected (Table 1).

Coagulase-negative Staphylococcus ST35 showed antimicrobial activity against seven out of 12 (58.3%) methicillin-resistant and six out of 11 (54.5%) methicillin-susceptible staphylococci. In addition, Staphylococcus ST35 showed the widest range of action, inhibiting the growth of other bacterial genera used as indicators, such as Lactobacillus, Pediococcus, Bacillus, Listeria and Enterococcus (Table 1). ST35 inhibited the growth of 37 out of 58 indicators used that belonged to six of the eight genera tested and to 15 of the 23 different species analyzed (Table 1). The bacteriocin activity was not demonstrated after filtration (Millipore, 45 mm) due to its adsorption to the cell membrane. Purification to homogeneity of the bacteriocin from ST35 and further characterization are currently under investigation.

In conclusion, some staphylococcal isolates, including methicillin-resistant staphylococci and other genera, produce bacteriocin with a wide spectrum of activity. Such bacteriocins could lead to a strong advantage toward adaptation for these producer strains in a mixed population medium, and could play a considerable role in the infection process.

Correspondence: Carmen Torres, Área Bioquímica y Biología Molecular, Universidad de La Rioja, Madre de Dios 51, 26006 Logroño, Spain.
Tel: 941 299750 Fax: 941 299721. E-mail: [email protected]

REFERENCES

1. Klaenhammer, T.R. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol Rev 1993; 12: 39-86.

2. Crupper, S.S., Iandolo, J.J. Purification and partial characterization of a novel antibacterial agent (Bact 1829) produced by Staphylococcus aureus KS11829. Appl Environ Microbiol 1996; 62: 3171-3175.

3. Netz, D.J., Sahl, H.G., Marcelino, R., Dos Santos Nascimento, J., De Oliveira, S.S., Soares, M.B., Do Carmo de Freire Bastos, M., Marcolino, R. Molecular characterization of aureocin A70, a multi-peptide bacteriocin isolated from Staphylococcus aureus. J Mol Biol 2001; 311: 939-949.

4. Jetten, A.M., Vogels, G.D. Characterization and extrachromosomal control of bacteriocin production in Staphylococcus aureus. Antimicrob Agents Chemother 1973; 4: 49-57.

5. Nakamura, T., Yamazaki, N., Taniguchi, H., Fujimura, S. Production, purification and properties of a bacteriocin from Staphylococcus aureus. Infect Immunol 1983; 39: 609-614.

6. Jetten, A.M., Vogels, G.D., de Windt, F. Production and purification of a Staphylococcus epidermidis bacteriocin. J Bacteriol 1972; 112: 235-242.

7. Nakamura, T., Hirai, K., Shibata, Y., Fujimura, S. Purification and properties of a bacteriocin of Staphylococcus epidermidis isolated from dental plaque. Oral Microbiol Immunol 1998; 13: 387-389.

8. Gamon, M.R., Moreira, E.C., de Oliveira, S.S., Teixeira, L.M., Bastos, M.D.C. Characterization of a novel bacteriocin-encoding plasmid found in clinical isolates of Staphylococcus aureus. Antonie Van Leeuwenhoek 1999; 75: 233-243.

9. Iqbal, A., Ahmed, S., Abid Ali, S., Rasool, S.A. Isolation and partial characterization of Bac2Ol: A plasmid-associated bacteriocin-like inhibitory substance from Staphylococcus aureus AB201. J Basic Microbiol 1999; 39: 325-336.

10. Navarro, L., Zarazaga, M., Sáenz, J., Ruiz-Larrea, F., Torres, C. Bacteriocin production by lactic acid bacteria isolated from Rioja red wines. J Appl Microbiol 2000; 88: 44-51.