Staphylococcus is a genus widely distributed in the environment and comprises important members of the major bacterial communities colonizing the skin and mucous membranes of humans and animals (Kranjec et al., 2020). This genus comprises a high diversity of species, traditionally separated into two major groups based on their ability to clot the plasma: coagulase-positive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS). CoNS is the broadest group, with more than 80% of species (Becker et al., 2015).

Although staphylococci often maintain a commensal or symbiotic relationship with their hosts, staphylococcal infections could develop in specific cases (de Freire Bastos et al., 2020). Some staphylococcal species are more related to human and animal infectious diseases, being Staphylococcus aureus one of the most important.

Individual bacterial strains living in highly competitive and polymicrobial environments have developed multiple types of interaction and self-defense mechanisms. The survival success can include different capabilities such as a higher specificity to a limited number of host attachment sites, a better ability to take up and metabolize micronutrients, or the production of antibacterial substances that inhibit competitors (Janek et al., 2016). The most widely distributed microbial defense mechanisms are bacteriocins, in most cases ribosomally synthesized peptides with antibacterial properties (França et al., 2021). From an ecological point of view, bacteriocins are synthesized to confer a selective advantage to the producer in terms of niche colonization ability since these molecules often display activity against closely related bacterial species (Heilbronner et al., 2021).

Staphylococci are no exception with respect to antimicrobial substances production. There is a wide variety of staphylococcal bacteriocins, highlighting the lantibiotic group (Class I), characterized by their post-translationally modifications, usually by enzymatic tailoring (Heilbronner et al., 2021). Moreover, other well-described antimicrobial peptides include those that remain unaltered (Class II bacteriocins), the heat-labile proteins differentiated between lytic and non-lytic bacteriocins (Class III) (Heng et al., 2007), and the cyclic molecules (Class IV) (de Freire Bastos et al., 2020; Newstead et al., 2020). Other antimicrobial peptides have recently been discovered in staphylococci isolates, such as those included in the sactipeptide group (de Freire Bastos et al., 2020) and the well-known fibupeptide lugdunin (a major non-ribosomal peptide, NRP) described by Zipperer et al. (2016). Moreover, bacteriocins previously described in other genera, such as the thiopeptide Micrococcin P1, have been also detected in staphylococci (Carnio et al., 2000). In addition, there is another type of antimicrobial substance known as bacteriocin-like inhibitory substances (BLIS), which are not obtained in pure form or fully characterized, and they have also been reported in staphylococci (James and Tagg, 1991; Mak, 2018).

Staphylococcus aureus is considered the most relevant CoPS in terms of bacteriocin production (generally, class II type bacteriocins), although CoNS bacteriocins (mostly lantibiotics) have been also reported in the literature (Bastos et al., 2009). Bacteriocin produced by staphylococci are active against pathogenic staphylococci, including methicillin-resistant S. aureus (Kranjec et al., 2020). These antimicrobial compounds could be of interest as food additives, therapeutic agents, or modulators of the microbiota (Soltani et al., 2021).

Due to the growing interest in the identification and characterization of new antimicrobial agents, this study sought to determine the production of BLIS in a large collection of CoNS and CoPS isolates of different origins (human, animal, food, and the environment) and to characterize the BLIS-producer (BLIS⁺) isolates at the genomic level to determine the presence of bacteriocin encoding genes as well as antimicrobial resistance (AMR) and virulence genes.

Antimicrobial resistance is a relevant health problem worldwide that needs the development of new strategies. The human–animal–environment interface is currently being considered under the OneHealth approach to better understand the ecology and dissemination of AMR and to control the silent pandemy, as is considered by some authors (Swift et al., 2019; Penna et al., 2021). In this context, the use of probiotics or new antimicrobial compounds, such as bacteriocins, is an interesting alternative to the use of conventional antibiotics. These peptides might become important biological weapons, especially against emerging drug-resistant bacteria, and they might be used in different areas, such as the food industry, medicine, veterinary, and agriculture (de Freire Bastos et al., 2020).

The Staphylococcus genus has been widely described in the literature as a bacteriocin producer. Several studies have described staphylococcins in strains from food products, such as milk (Hyicin 3682, Aureocin A70, and Aureocin A53) (Netz et al., 2001; Netz et al., 2002; Carlin Fagundes et al., 2017), fermented food (Micrococcin P1 and Nukacin ISK-1) (Cundliffe and Thompson, 1981; Sashihara et al., 2000), fish (Nukacin KQU-131) (Wilaipun et al., 2008), livestock (BacCH91 and Gallidermin) (Kellner et al., 1988; Wladyka et al., 2013), and also in strains from pets (BacSp222) (Wladyka et al., 2015). However, very few studies have reported bacteriocins produced by staphylococci from wild animals, except for some studies on animals from the marine environment (Prichula et al., 2021). Regarding the available information about the environment, the soil is the most widely studied natural source of antimicrobial peptides, and Bacillus is the most representative genus of soil bacteriocin producers (Zimina et al., 2020).

In humans, bacteriocin production has been detected in clinical and commensal Staphylococcus isolated from the skin (Capidermicin, NisinJ, and Staphylococcin C55) (Dajani and Wannamaker, 1969; Lynch et al., 2019; O’Sullivan et al., 2020) and nose (Nukacin IVK45 and Lugdunin) (Janek et al., 2016; Zipperer et al., 2016).

In this study, production of BLIS was found in 6.7% of isolates of a large collection of staphylococci (both CoPS and CoNS) of many different origins (wild animal, food, pet, environment, and human) that were tested against a wide series of indicator bacteria, including MDR bacteria, and also bacteria with relevant mechanisms of AMR or with zoonotic interest. According to our results, the species with the highest percentages of BLIS⁺ isolates were S. chromogenes (38.5%), S. pseudintermedius (26.7%), and S. warneri (23.1%), which highlights the capacity of CoNS, usually considered as common colonizers and non-pathogenic bacteria, to compete against other bacteria, including pathogens (Janek et al., 2016; O’Sullivan et al., 2019). The percentages of BLIS⁺ isolates in other staphylococcal species were lower: S. hyicus (16.7%), S. hominis (12.5%), S. epidermidis (10.5%), S. xylosus (6.25%), S. sciuri (6.1%), and S. aureus (3.4%). S. epidermidis has been described as one of the most frequent bacteriocin-producing CoNS species, mainly among human isolates (Bastos et al., 2009; Janek et al., 2016). Although all human S. epidermidis isolates tested in our study were BLIS-negative, S. epidermidis was the fifth CoNS species with higher antimicrobial activity rates in our study, although all the BLIS⁺ S. epidermidis isolates were recovered from food.

Some authors consider bacteriocin production to be a common characteristic of bacteria since most of them can produce these antimicrobial peptides (Cotter et al., 2005). Bacteriocin production has been reported in the literature for CoPS and CoNS, and S. aureus and S. epidermidis have been highlighted as highly prevalent producer species or at least with the better-characterized bacteriocins (Bastos et al., 2009).

Statistically significant differences were detected in this study between the origin of the isolates and the production of BLIS. Considering the total collection of Staphylococcus tested, BLIS production was more frequently detected among isolates of pets (30%), food (6.1%), and wild animals (6.2%) (percentages obtained with respect to the number of total isolates tested in each origin); these BLIS⁺ isolates represented 25%, 28.3%, and 41.7% of the 60 BLIS⁺ isolates, respectively. The high rate of BLIS production detected among isolates of pets might be explained by the fact that all the isolates analyzed from this originally belonged to the species S. pseudintermedius, one of the higher BLIS⁺ species detected in our study, as previously indicated. In humans, only one of the 27 tested isolates (3.7%) showed BLIS production, and this isolate was S. pseudintermedius, which interestingly was obtained from the clinical sample of a human cohabitating with pets. Higher frequencies of bacteriocin production have been previously reported in nasal staphylococci of human origin (80%) when bacteria of the nasal ecosystem were used as indicator bacteria (Janek et al., 2016). A wide diversity of variables could explain the differences observed in bacteriocin production rates, but the different origins and species of the staphylococci tested for and the different indicator bacteria employed might be involved.

Most of the known bacteriocins have a narrow spectrum of activity, often active against closely related bacteria (Cotter et al., 2013); nevertheless, in relation to staphylococcins, a wide variety of antimicrobial profiles against pathogens have been described depending on the peptide (Streptococcus, Enterococcus, Corynebacterium, Bacillus, Clostridioides, Klebsiella, and Neisseria, among others) (de Freire Bastos et al., 2020). This might indicate that this genus needs bacteriocins as a strategy to be maintained in complex ecosystems in different hosts (Janek et al., 2016).

All Gram-positive indicator bacteria were inhibited by at least one of the BLIS⁺ staphylococci, but this did not happen with Gram-negative indicator bacteria. It was especially relevant to the antimicrobial activity detected against S. delphini, S. pseudintermedius, C. perfringens, and M. luteus. Notably, C. perfringens is an important pathogen in poultry production (Nhung et al., 2017), and S. pseudintermedius is an emerging zoonotic pathogen in humans, especially in those with close contact with dogs/cats (Lozano et al., 2017).

Interestingly, we detected 7 BLIS⁺ isolates of the species S. aureus, S. pseudintermedius, S. sciuri, and S. hominis, which presented antimicrobial activity against > 70% of the indicator bacteria, including MRSP, MRSA, VRE, L. monocytogenes, C. perfringens, or S. suis, among others. These BLIS⁺ isolates are of interest for further in-depth characterization.

It is known that staphylococcal strains can carry one or more gene clusters responsible for bacteriocin production (de Freire Bastos et al., 2020). In our study, 16 of the 60 BLIS⁺ isolates showed one of the bacteriocin genes analyzed. Many Staphylococcus isolates are producers of lantibiotics, and a variety of these bacteriocins have been described (Neubauer et al., 1999). Significantly, putative lantibiotic-biosynthetic gene clusters have been recently found in the genome of S. capitis, which share homology with the biosynthetic systems of epidermin/gallidermin and the non-lantibiotic bacteriocin epidermicin (Kumar et al., 2017). According to our results, structural genes encoding putative lantibiotic-like bacteriocins were detected by PCR and sequencing in 11 isolates, both CoNS (n = 5) and CoPS (n = 6). Moreover, the gene of the recently described bacteriocin BacSp222 (Wladyka et al., 2015) was detected in three of the BLIS⁺ S. pseudintermedius isolates, and interestingly, all showed H-Act; nevertheless, this gene was not found in the other staphylococci species tested. In addition, it is worth highlighting the detection of the gene encoding a putative circular bacteriocin (uberolysin-like) in two of our S. chromogenes BLIS⁺ isolates tested. The circular bacteriocins have been commonly described in Bacillus and have been rarely identified in Staphylococcus species, excepting the Aureocyclin 4185 described in S. aureus (Potter et al., 2014). The detection of this putative circular bacteriocin in two S. chromogenes isolates would be the first report of this CoNS species, indicating the possible transfer of genetic material between staphylococcal species or the possible detection of a new bacteriocin. Notably, four of our isolates with high (X3041, X3011, and C5835) or medium (X2969) antimicrobial activity lacked all the 23 bacteriocin encoding genes tested. Further studies will be performed with these isolates to determine if they produce new bacteriocins that could be of interest.

According to the AMR phenotype and genotype and the virulence content of the BLIS⁺ isolates, it was observed in this study that 30.7% of BLIS⁺ CoNS and 19% of CoPS were susceptible to all antibiotics tested; nevertheless, 12.8% and 33.3% of BLIS⁺ CoNS and CoPS isolates, respectively, were MDR. Moreover, all the CoNS lacked the virulence genes tested, while different virulence gene profiles were observed among CoPS. Currently, the use of bacteriocins or bacteriocin-producing bacteria as an alternative to antibiotics needs to consider strains that meet all safety, efficacy, and viability criteria to be used in consortium formulations, which are being proposed as the next generation of probiotics (Eveno et al., 2021). In this context, isolates lacking acquired AMR genes or virulence genes are of special relevance.

Bacteriocin production is a defense strategy or adaptive mechanism that contributes to the success of niche colonization. In this study, 6.7% of the staphylococci tested were BLIS⁺, showing a wide variety of inhibition patterns in relation to the species of producer isolates and their origins. It is worth noting the interest of CoNS isolates, due to their high antimicrobial activity profiles confirmed by the detection of bacteriocin encoding genes and the lack of relevant acquired AMR or virulence genes. These results leave a gateway for further biochemical and genetic characterization of high relevance for these BLIS⁺ isolates that can be considered as excellent candidates for a further in-depth study. To conclude, the OneHealth approach is important to better understand the interactions between colonizing bacteria in specific niches and their spread to other environments, considering the molecular ecology of AMR and other mechanisms of bacterial fitness.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

CT, MZ, and CL contributed to the design and the general supervision of the study. RF-F developed most of the experimental work and the first version of the manuscript. CT made the first revision of the manuscript. PE, LR-R, and IA contributed to some experimental laboratory work related to AMR genes and molecular typing. CT and MZ contributed to project funding. All authors revised the different versions of the manuscript, read, and agreed to the submitted version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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