Draft genome sequence of Staphylococcus agnetis 3682, the producing strain of the broad-spectrum lantibiotic agneticin 3682.

OBJECTIVES: Here we report the draft genome sequence of Staphylococcus agnetis 3682, a strain producing agneticin 3682, a broad-spectrum lantibiotic with potential medical applications. The inhibitory activity of S. agnetis 3682 against multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA) isolates involved in human infections was also investigated. METHODS: A sequencing library was constructed using a Nextera XT DNA Library Preparation Kit. An Illumina MiSeq system was used to perform whole-genome shotgun sequencing. De novo genome assembly was performed using the A5-miseq pipeline. Staphylococcus agnetis 3628 genome annotation was performed by the RAST server, and BAGEL4 and antiSMASH v.4.0 platforms were used for mining bacteriocin gene clusters. The inhibitory activity of S. agnetis 3682 against 20 multidrug-resistant MRSA strains involved in human infections in two Brazilian hospitals was determined by the deferred antagonism assay on brain-heart infusion (BHI) agar plates. RESULTS: The total scaffold size was determined to be 2 502 817bp with a G+C content of 35.6%. Genome analyses revealed 2437 coding sequences, 76 RNA genes, 27 genes involved in drug resistance and 2 bacteriocinogenic gene clusters (for agneticin 3682 and hyicin 4244). Staphylococcus agnetis 3682 inhibited 80% of the MRSA isolates tested. CONCLUSION: This study describes the main features of the draft genome of S. agnetis 3682, a strain producing the first bacteriocin (agneticin 3682) reported in this species. A second gene cluster encoding a sactipeptide was also found in the bacterial chromosome. Agneticin 3682 shows a new potential application against clinical MRSA isolates.

Hyicin 3682, a bioactive peptide produced by Staphylococcus hyicus 3682 with potential applications for food preservation.

Bacteriocins are peptides produced by bacteria and having inhibitory activity against other bacteria. Many of these substances may be useful as antibacterial agents for practical applications. In this study, 21 Staphylococcus spp. isolated from pigs, dogs and bovine milk in different states of Brazil were investigated for staphylococcin production. Hyicin 3682, a bacteriocin produced by one such strain, inhibited almost all strains tested, including Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus. PCR experiments showed that hyicin 3682 is lantibiotic-related, but not identical, to both epidermin and Bsa. The maximum production of hyicin 3682 (6,400 AU/ml) was observed after 24 h of growth in BHI medium at 37 °C. Hyicin 3682 proved to be a cationic, small antimicrobial peptide with a molecular mass of 2,139 Da. It exhibited resistance to low pH and to heating at 65 °C, and partial sensitivity to proteolytic enzymes. Taken together, these results suggest that hyicin 3682, the first bacteriocin characterized in Staphylococcus hyicus, has potential biotechnological applications as a food preservative. Moreover, hyicin 3682 was able to inhibit its producer strain, suggesting that an effective immune system for specific protection against hyicin 3682 is not found in its producer strain, a characteristic not described thus far for other staphylococcins.

Aureocins 4185, bacteriocins produced by Staphylococcus aureus 4185: potential application in food preservation.

In the present study, the bacteriocins produced by Staphylococcus aureus 4185, a strain isolated from bovine mastitis, were purified and partially characterized. After purification by ammonium sulfate precipitation, cation-exchange chromatography, and five runs of high-performance liquid chromatography (HPLC), antimicrobial activity was recovered with 40% and 80% isopropanol, suggesting that more than one antimicrobial peptide, named aureocins 4185, is produced by S. aureus 4185. Mass spectrometry analyses revealed three peptides eluted with 40% isopropanol: peptide A (2,305.3 +/-1.5 Da), peptide B (2,327.3 +/-1.5 Da), and peptide C (3,005.5 +/-1.5 Da), and two peptides eluted with 80% isopropanol: peptide D (6,413.5 +/-1.5 Da) and peptide E (12,834.5 +/-1.5 Da). Although five peptides have been detected, only four small peptide sequences were obtained by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)/TOF mass spectrometry analyses: SLLEQFTGK (eluted with 40% isopropanol), ALLYDER, NNTSHNLPLGWFNVK, and NNLAQGTFNATK (eluted with 80% isopropanol). The sequences SLLEQFTGK and ALLYDER revealed identity with hypothetical peptides with unknown function. The sequences NNTSHNLPLGWFNVK and NNLAQGTFNATK showed similarity to a segment of a precursor of staphylococcal autolysins. The antimicrobial activity detected in the supernatant of strain 4185 proved to be resistant to heat treatment at 65°C; however, treatment at 80°C abolished completely its antimicrobial properties. The concentrated supernatant containing aureocins 4185 exhibited a strong bacteriolytic activity toward Micrococcus luteus ATCC 4698. Additionally, aureocins 4185 exhibited antagonistic activity against important foodborne pathogens, including Listeria monocytogenes, thus showing a potential application in food preservation.

Activity of staphylococcal bacteriocins against Staphylococcus aureus and Streptococcus agalactiae involved in bovine mastitis.

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

Production of bacteriocins by coagulase-negative staphylococci involved in bovine mastitis.

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.