From raw milk we found 10 Lactococcus garvieae isolates that produce a new broad-spectrum bacteriocin. Though the isolates were obtained from different farms, they turned out to possess identical inhibitory spectra, fermentation profiles of sugars, and repetitive sequence-based PCR (rep-PCR) DNA patterns, indicating that they produce the same bacteriocin. One of the isolates ( L. garvieae KS1546) was chosen for further assessment. Purification and peptide sequencing combined with genome sequencing revealed that the antimicrobial activity was due to a bacteriocin unit composed of three similar peptides of 32 to 34 amino acids. The three peptides are produced without leader sequences, and their genes are located next to each other in an operon-like structure, adjacent to the genes normally involved in bacteriocin transport (ABC transporter) and self-immunity. The bacteriocin, termed garvicin KS (GarKS), showed sequence homology to four multipeptide bacteriocins in databases: the known staphylococcal aureocin A70, consisting of four peptides, and three unannotated putative multipeptide bacteriocins produced by Bacillus cereus . All these multipeptide bacteriocin loci show conserved genetic organization, including being located adjacent to conserved genetic determinants (Cro/cI and integrase) which are normally associated with mobile genetic elements or genome rearrangements. The antimicrobial activity of all multipeptide bacteriocins was confirmed with synthetic peptides, and all were shown to have broad antimicrobial spectra, with GarKS being the most active of them. The inhibitory spectrum of GarKS includes important pathogens belonging to the genera Staphylococcus , Bacillus , Listeria , and Enterococcus . IMPORTANCE Bacterial resistance to antibiotics is a very serious global problem. There are no new antibiotics with novel antimicrobial mechanisms in clinical trials. Bacteriocins use antimicrobial mechanisms different from those of antibiotics and can kill antibiotic-resistant bacteria, but the number of bacteriocins with very broad antimicrobial spectra is very small. In this study, we have found and purified a novel three-peptide bacteriocin, garvicin KS. By homology search, we were able to find one known and three novel sequence-related bacteriocins consisting of 3 or 4 peptides. None of the peptides has modified amino acids in its sequence. Thus, the activity of all bacteriocins was confirmed with chemically synthesized peptides. All of them, especially garvicin KS, have very broad antibacterial spectra, thus representing a great potential in antimicrobial applications in the food industry and medicine.

The emergence of antibiotic-resistant pathogens has caused a serious worldwide problem in infection treatment in recent years. One of the pathogens is methicillin-resistant Staphylococcus aureus (MRSA), which is a major cause of skin and soft tissue infections. Alternative strategies and novel sources of antimicrobials to solve antibiotic resistance problems are urgently needed. In this study, we explored the potential of two broad-spectrum bacteriocins, garvicin KS and micrococcin P1, in skin infection treatments. The two bacteriocins acted synergistically with each other and with penicillin G in killing MRSA in vitro. The MICs of the antimicrobials in the three-component mixture were 40 ng/ml for micrococcin P1 and 2 μg/ml for garvicin KS and penicillin G, which were 62, 16, and at least 1,250 times lower than their MICs when assessed individually. To assess its therapeutic potential further, we challenged the three-component formulation in a murine skin infection model with the multidrug-resistant luciferase-tagged MRSA Xen31, a strain derived from the clinical isolate S. aureus ATCC 33591. Using the tagged-luciferase activity as a reporter for the presence of Xen31 in wounds, we demonstrated that the three-component formulation was efficient in eradicating the pathogen from treated wounds. Furthermore, compared to Fucidin cream, which is an antibiotic commonly used in skin infection treatments, our formulation was also superior in terms of preventing resistance development.

Bacterial infections in dairy cows are a major burden to farmers worldwide because infected cows require expensive treatments and produce less milk. Today, infected cows are treated with antibiotics, a practice that is becoming less effective due to antibiotic resistance.

Listeria monocytogenes is an important food-borne pathogen and a serious concern to food industries. Bacteriocins are antimicrobial peptides produced naturally by a wide range of bacteria mostly belonging to the group of lactic acid bacteria (LAB), which also comprises many strains used as starter cultures or probiotic supplements. Consequently, multifunctional strains that produce bacteriocins are an attractive approach to combine a green-label approach for food preservation with an important probiotic trait. Here, a collection of bacterial isolates from raw cow’s milk was typed by 16S rRNA gene sequencing and MALDI-Biotyping and supernatants were screened for the production of antimicrobial compounds. Screening was performed with live Listeria monocytogenes biosensors using a growth-dependent assay and pHluorin, a pH-dependent protein reporting membrane damage. Purification by cation exchange chromatography and further investigation of the active compounds in supernatants of two isolates belonging to the species Pediococcus acidilactici and Lactococcus garvieae suggest that their antimicrobial activity is related to heat-stable proteins/peptides that presumably belong to the class IIa bacteriocins. In conclusion, we present a pipeline of methods for high-throughput screening of strain libraries for potential starter cultures and probiotics producing antimicrobial compounds and their identification and analysis.

The rise of antibiotic-resistant bacteria is among the biggest challenges in human and veterinary medicine. One of the major factors that contributes to resistance is use of frontline clinical antibiotics in veterinary practices. To avoid this problem, searching for antimicrobials aimed at veterinary applications is becoming especially important. Thiopeptide micrococcin P1 and leaderless peptide EntEJ97s are two different bacteriocins that are very active against many gram-positive bacteria; however, sensitive bacteria can rapidly develop resistance towards those bacteriocins. To overcome this problem, we searched for synergy between those bacteriocins and conventional antibiotics against methicillin-resistant Staphylococcus pseudintermedius (MRSP): a common pathogen in animal skin infections. The two bacteriocins acted synergistically with each other and with penicillin G against MRSP clinical isolates in both planktonic and biofilm assays; they also prevented resistance development. The therapeutic potential was further validated in a murine skin infection model that showed that a combination of micrococcin P1, EntEJ97s and penicillin G reduced cell-forming units of MRSP by 2-log₁₀ CFU/g. Taken together, our data show that a combination of bacteriocins with conventional antibiotics can not only prevent resistance development but also pave the way to revitalize some old, less useful antibiotics, such as penicillin, which by itself has no effect on methicillin-resistant pathogens.

The leaderless bacteriocin Garvicin KS (GarKS) is a potent antimicrobial, being active against a wide range of important pathogens. GarKS production by the native producer Lactococcus garvieae KS1546 was however relatively low (80 BU/ml) under standard laboratory growth conditions (batch culture in GM17 at 30°C). To improve the production of GarKS, we systematically evaluated the impact of different media and media components on bacteriocin production. Based on the outcomes a new medium formulation was made to greatly improve bacteriocin production. The new medium composed of pasteurized milk and tryptone (PM-T), increased GarKS production about 60-fold compared to that achieved in GM17. GarKS production was increased further 4-fold (i.e., to 20,000 BU/ml) by increasing gene dose of the bacteriocin gene cluster ( gak ) in the native producer. Finally, a combination of the newly composed medium (PM-T), an increased gene dose and a cultivation at a constant pH 6 and a 50-60% dissolved oxygen level in growth medium, gave rise to a GarKS production of 164,000 BU/ml. This high production, which is about 2000-fold higher compared to that initially achieved in GM17, corresponds to a GarKS production of 1.2 g/L. To our knowledge, this is one of the highest bacteriocin production reported hitherto. Importance: Low bacteriocin production is a well-known bottle-neck in developing bacteriocins into large-scaled and useful applications. The present study shows different approaches that significantly improve bacteriocin production. This is an important research field to better exploit the antimicrobial potential of bacteriocins, especially with regard to the decreasing effect of antibiotics in infection treatments due to the global emergence of antibiotic resistance.