Isolation of Bacteriocin-producing Staphylococcus spp. Strains from Human Skin Wounds, Soft Tissue Infections and Bovine Mastitis

Bacterial cells may produce several types of substances that inhibit the growth of other microorganisms. These antimicrobial agents belong to different classes of molecules, ranging from the simple compounds, like hydrogen peroxide or lactic acid to more complicated: antibiotics, peptides, exotoxins or lytic enzymes (Brito et al., 2011). Among them, the agents of proteinaceous nature, peptides and proteins, seem to be especially interesting. Bacteria produce two essential classes of products of this type: bacteriocins and peptide antibiotics. Both of them are secreted to help the host strain to eliminate other microorganisms from the place of its growth. However, there are some important differences between these two classes of agents. First of all, enzymes synthesize the peptide antibiotics, whilst the bacteriocins are ribosomally synthesized. Bacteriocins are targeted at a narrow spectrum of bacteria often within the species of the producer or closely related ones, while the classical antibiotics are active against broad spectra of bacteria. Another important feature that separates bacteriocins from antibiotics is their potency against susceptible bacteria; bacteriocins are unique because they can kill bacteria at nanomolar concentrations, while antibiotics are needed in much higher concentrations (Nes, 2011). The bacteriocins are included in the larger group of antimicrobial substances defined as antimicrobial peptides (AMPs). These peptides are ribosomally synthesized by all domains of living organisms (eukaryotes, bacteria and archaea). Similarly as bacteria the other organisms (e.g. plants, animals and humans) produce AMPs for elimination of pathogenic microorganisms and prevention of infections (AMPs constitute an important element of their innate immune system) (Rashid et al., 2016). AMPs such as human defensins, human neutrophil peptides and many bacterial bacteriocins are cationic and are able to bind to anionic parts of bacterial surface (e.g. anionic lipids or other anionic components) (Joerger, 2003). AMP’s could undergo posttranslational modification e.g. proteolytic processing or glycosylation (Rashid et al., 2016).

The modes of action of AMPs, including most of the bacteriocins, involve interactions with the bacterial cell membrane, and forming complexes with lipid II – a highly conserved precursor of the bacterial cell wall (Malanovic and Lohner, 2016). Cell membrane permeabilization leads to efflux of important intracellular molecules such as mono- and divalent ions (K⁺, Na⁺, Ca²⁺ and Mg²⁺) and ATP (Di Meo et al., 2016; Yeaman and Yount, 2003). Binding the peptides with lipid II inhibits peptidoglycan synthesis and additionally promotes pore formation and membrane disruption (Malanovic and Lohner, 2016). Structure-activity analyses of a broad range of peptides showed two main requirements for their antimicrobial activity: (1) a cationic charge and (2) an induced amphipathic conformation. The diversity of lipids among pathogens could explain the differences in activity of a single peptide between different types of microorganisms (Powers and Hancock, 2003).

Due to high selectivity and activity against a substantial array of pathogenic microorganisms, AMP’s are a promising alternative to conventional antibacterial antibiotics (Kosikowska and Lesner, 2016; Ołdak and Zielińska, 2017). Compared to conventional antibiotic therapy, killing of microorganisms by peptides is more rapid and they can ‘attack’ multiple molecular targets related to membrane and cell wall, which highly prevents development of resistant strains. However, the results of recent years revealed that many important Gram-positive as well as Gram-negative bacteria, including dangerous human and animal pathogens, have developed mechanisms of modification of most important molecular targets of AMPs, namely cell membrane and lipid II. As a consequence these strains are highly resistant to AMPs’ activity (Andersson et al., 2016).

In the present study, a collection of Staphylococcus spp. strains isolated from human skin wounds, soft tissues infections, as well as from milk of bovine suffering from mastitis (S. aureus, S. epidermidis, S. xylosus) was screened for their ability to produce antibacterials, particularly with proteinaceous structure, with activity against other staphylococci. This research was focused on identification and preliminary characterization of the newly discovered antimicrobial peptides in order to uncover their potential therapeutic application in medicine and veterinary science for elimination of pathogenic bacteria from food products and also possibilities of their large-scale production.

Screening of the 206 bacterial isolates resulted in the identification of six strains producing antistaphylococcal agents. Three producing strains were S. aureus isolated from bovine mastitis (assigned in the collection as 30, 31 and 32), one was S. aureus recovered from human skin wound (K1), one agent was produced by S. xylosus M2B and the last of producing strain was S. epidermidis 30c1. The antibacterial activity of three (produced by S. aureus 30, 31 and 32) out of six substances was not affected by the treatment with proteolytic enzymes, suggesting their non-proteinaceous structure. Further tests eliminated two additional strains (K1 and 30c1), since they produced antimicrobial substances exclusively when grown on solid (agar) medium while no antibacterial activity was detected in the supernatants collected from the suspension cultures. Therefore, completion of the screening procedure resulted in the identification of one strain effectively producing the antibacterial substance with protease-sensitive structure, i.e. potential bacteriocin or peptide antibiotic. This strain (M2B) and the produced agent were the subjects of further analysis.

Based on the sequence of the gene coding for 16S rRNA, the producing strain was identified as S. xylosus (99,9% of identity of this gene sequence with the sequences of 16S rRNA genes of other bacteria of the this genus) – labeled in the collection as M2B. The antimicrobial agent synthesized by this strain was efficiently produced both on solid as well as in a liquid medium (Fig. 1a and Fig. 2) and its’ activity was eliminated in the presence of proteases (Fig. 3).

Fig. 1.

Fig. 2.

Fig. 3.

The antimicrobial spectrum of the putative antimicrobial peptide produced by S. xylosus M2B was determined by the well overlay method. Growth inhibition zones were observed with 13 strains of staphylococci (including nine S. aureus, three S. xylosus and one S. epidermidis strains), L. monocytogenes and M. luteus (Table II). Diameters of the growth inhibitory zones were in the range of 12–25 mm; surprisingly apparent substantial differences in sensitivity to the putative AMPs were found among the different S. aureus strains.

Conditions for the effective production of the putative bacteriocin by S. xylosus M2B in a liquid culture were optimized in the terms of medium type, time period and incubation temperature. Comparison of the production efficiency in five different commercially available media, recommended for staphylococci culture, revealed that the optimal production of the putative AMP was observed in TSB medium. Slightly lower level of peptide production was found in MHB and LB media and no production was detected in CAA and BHI media (Fig. 2).

The maximum level of AMP production by S. xylosus M2B was observed after 24 h of incubation in TSB medium, and the optimal temperature was 37°C.

The tests of thermostability revealed that the putative antimicrobial peptide did not lose its activity after 15 minutes of heating at 90°C. After 30 min of incubation in 90°C peptide lost its activity (inhibition zone was significantly smaller).

Studies on the antimicrobial substances produced by staphylococci, have not been very extensive so far. However, there are a few peptides exhibiting antistaphylococcal activity produced by bacteria that belong to this genus, including both CNS – coagulase negative and CPS – coagulase positive staphylococci. The most promising examples of AMPs produced by CNS are the following bacteriocins: Pep5, epicidin 280, epilancin K7, epidermin, nukacin ISK-1, simulancin 3299, and the most extensively characterized bacteriocins produced by CPS are: staphylococcin C55, aureocin A70, aureocin A53 (Nascimento et al., 2006; Varella Coelho et al., 2007). The detailed characteristics of the best characterized staphylococcins has been presented in the review by Bastos and coworkers (Bastos et al., 2009). The authors also emphasized that staphylococcins may be applied, solely or in a combination with other chemical agents. The use of combinations of antimicrobials is common in the clinical setting, and the most important advantages coming from such approach are: 1) expanding the spectrum of organisms that can be targeted, 2) prevention of the emergence of resistant organisms, 3) decreasing of toxicity by allowing lower doses of both agents (Kurlenda and Grinholc, 2012).

Our studies, results of which are presented in this work, are probably one of the very few examples of an extensive screening aimed at the identification of antimicrobial peptides produced by staphylococci isolated from humans (skin and soft tissue infections n = 48) or animals (strains isolated from bovine with mastitis n = 158). From the set of 206 strains, we identified six strains producing any antimicrobials (2.9%), of which, one was confirmed to be a putative peptide or protein with antimicrobial activity, efficiently produced in a liquid medium. Two out of three antimicrobial – producing strains identified in these studies did not produce the antimicrobial agents at the detectable level when grown in liquid media. Similar problems were recently observed by Braem and coworkers (2014), who identified Staphylococcus chromogenes L217 strain (isolated from teat apices of dairy cows) producing nukacin-like bacteriocin displaying a broad spectrum of antimicrobial activity. Unfortunately, the bacteriocin was only produced in significant quantities when the producer strain was grown on solid agar medium.

The molecular structure of the putative AMP produced by S. xylosus M2B is not known until now. This work is in progress in our laboratory, but preliminary results indicate that it could be a novel, previously undescribed compound. Its antimicrobial spectrum is interesting, since it is relatively narrow. Additionally, the good thermostability is advantageous, which could be especially important in the case of potential application of the identified antimicrobial peptide for the elimination of staphylococci from food products, but also for purification procedure, e.g. preliminary separation of the peptide from other components of the cell free after-culture supernatant by the heat treatment.

The produced peptide exhibits also strong activity against other important foodborne pathogenic bacteria L. monocytogenes. Thus, it could be also applied for protection of food products against these bacteria.

The recent studies carried out by Hewelt-Belka and coworkers (2016) revealed some important differences in the lipid composition of S. aureus clinical isolates. The observed, by the mentioned authors, differences in the lipid patterns between sensitive and resistant S. aureus strains suggest that antibiotic susceptibility may be associated with the lipid composition of bacterial cells. The lipids that were found to significantly differ between antibiotic-resistant and antibiotic-sensitive clinical isolates are involved in the biosynthesis of major S. aureus membrane lipids and lipoteichoic acid. The research of Powers and Hancock (2003) revealed that the lipid composition of the bacterial cell membrane is crucial for AMPs activity. Thus the observed in our studies differences in the susceptibility of Staphylococcus spp. strains tested to the agent produced by M2B can be caused by some differences in the lipid composition of their cell membranes.

In our studies, the prevalence of isolation of strains producing AMPs was found to be at a relatively low level – below 2% (three out of 206 isolates), and only one strain (prevalence below 0.5%) produced a peptide that is worthy of further studies. However, we do not have any doubts that staphylococci represents an interesting and promising source of uncharacterized antimicrobial peptides, which could be used for treatment or prophylaxis of infections caused by other bacteria that belong to the Staphylococcus spp. but also other important pathogenic microorganisms e.g. L. monocytogenes. Unfortunately, the research in this field is limited. Each year, numerous publications concerning the antibiotic resistance of the staphylococci isolated from both, human and animal (especially bovine mastitis) infections from different world regions are presented. In the case of some of these publications, large populations, counting even several hundreds of isolates, are investigated. Unfortunately, very few of these isolates are investigated for their potential production of antimicrobial agents. The methods used for screening the ‘producing strains’ are relatively easy and could be performed in most of microbiological laboratories. Thus, we encourage other research groups, which have access to the large number of human or animal isolates of staphylococci, for investigation of the production ability of potential antimicrobial peptides by these strains.