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Article

Bacteriocin-Producing Staphylococci and Mammaliicocci Strains for Agro-Food and Public Health Applications with Relevance of Micrococcin P1

by
Rosa Fernández-Fernández
1,
Carmen Lozano
1,
Allelen Campaña-Burguet
1,
Carmen González-Azcona
1,
Tamara Álvarez-Gómez
1,
Rocío Fernández-Pérez
2,
Raquel Peña
3,
Myriam Zarazaga
1,
Jaime Carrasco
4 and
Carmen Torres
1,*
1
Area of Biochemistry and Molecular Biology, OneHealth-UR Research Group, University of La Rioja, 26006 Logroño, Spain
2
Instituto de Ciencias de la Vid y del Vino (ICVV) (Universidad de La Rioja, Consejo Superior de Investigaciones Científicas (CSIC), Gobierno de La Rioja), 26007 Logroño, Spain
3
Department of Microbiology and Parasitology, Instituto de Investigación Sanitaria de Navarra (IdiSNA), University of Navarra, 31008 Pamplona, Spain
4
Department Ecology of Cultivated Mushrooms, Regional Institute for Agri-Food and Forest Research and Development (IRIAF), 16194 Cuenca, Spain
*
Author to whom correspondence should be addressed.
Antibiotics 2025, 14(1), 97; https://doi.org/10.3390/antibiotics14010097
Submission received: 28 November 2024 / Revised: 28 December 2024 / Accepted: 5 January 2025 / Published: 16 January 2025
(This article belongs to the Special Issue Editorial Board Members’ Collection Series: New Strategies to Overcome Antibiotic Resistance)
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Abstract

:
Antimicrobial-producing strains and their bacteriocins hold great promise for the control of bacterial diseases, being an attractive alternative to antibiotics. Thus, the aim of this study was to evaluate the inhibitory activity of 15 bacteriocin-producing staphylococci and mammaliicocci (BP-S/M) strains and their pre-purified extracts with butanol (BT) against a collection of 27 harmful or zoonotic strains (including Gram-positive/-negative bacteria and molds) with relevance in the public health and agro-food fields. These indicators (excluding Gram-negative strains) were grouped into seven categories based on their potential application areas: dairy livestock mastitis, avian pathogen zoonoses, swine zoonoses, food safety, aquaculture, wine making, and mushroom cultivation. In addition, cross-immunity assays between the BP-S/M strains were carried out to identify potential strain combinations to enhance their activity against pathogens. Finally, the hemolytic and gelatinase activities were tested in the BP-S/M strains. A strong inhibitory capacity of the BP-S/M strains was verified against relevant Gram-positive indicators, such as methicillin-resistant Staphylococcus aureus, Listeria monocytogenes, and Clostridium perfringens, among others, while no activity was detected against Gram-negative ones. Interestingly, several BT extracts inhibited the two mold indicators included in this study as representants of mushroom pathogens. The Micrococcin P1 producer Staphylococcus hominis C5835 (>60% of indicators were intensively inhibited by all the methods) can be proposed as a potential candidate for the control of bacterial diseases in the aforementioned categories alone or in combination with other BP-S/M strains (mainly with Staphylococcus warneri X2969). In this regard, five potential combinations of BP-S/M strains that enhanced their activity against specific pathogens were detected.

1. Introduction

The emergence of antibiotic resistance in commensal and pathogenic bacteria, due to the high use of antibiotics in humans and animals, has become an unresolved challenge to public health worldwide that needs to be addressed. In this respect, natural antimicrobial agents, including bacteriocins, have attracted extensive attention as a new microbial barrier in both the food and veterinary sectors [1]. Moreover, under the One Health approach, it is important to expand the study not only with human bacterial pathogens but also with those that cause severe diseases in animals and that affect the production of livestock industries (bovine, avian, or swine) with important economic activities worldwide [2,3].
Among the most relevant human opportunistic pathogens are Escherichia coli and Staphylococcus aureus [4], especially methicillin-resistant isolates (MRSA), which have been reported as an important target in both community and hospital settings, causing skin, wound, bloodstream, and other types of infections [5,6,7]. Moreover, these bacteria and other harmful microorganisms are also relevant in livestock production, highlighting Streptococcus suis in swine, Clostridium perfringens in poultry, and other Gram-positive/Gram-negative (G-positive/G-negative) pathogens engaged in bovine infections such as S. aureus, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus agalactiae, E. coli, Klebsiella pneumoniae, and Mycoplasma spp., among others. Concretely, S. aureus is responsible for about 50% of mastitis cases, and since this bacterium can inhabit the mammary gland and form abscesses, it is more difficult to combat upon using antibiotics [8,9]. In addition, S. aureus can also cause frequent problems in the food industry leading to important economic losses [10].
Potential alternatives to limit the use of antibiotics in the agro-food sector and in public health have been proposed, including antimicrobial peptides, bacteriophages, nanomedicines, probiotics, phytochemicals, and photodynamic light therapy, among others [11,12,13,14]. Bacteriocins are antimicrobial peptides produced by bacteria, mainly of ribosomal synthesis, with activity predominantly directed against bacteria closely related to the producing strain. There is a great diversity of bacteriocins, which can typically be classified into three classes based on their size, microbial target, mode of action, release, and immunity mechanisms. In addition, bacteriocins have different modes of action, the formation of pores in the target cell membrane being one of the most frequent. Commonly, bacteriocins are classified as Class I, post-transcriptionally modified and very diverse (lanthipeptides, head-to-tail cyclized peptides, and thiopeptides, among others); Class II, small and heat-stable; or Class III, large and thermally unstable peptides [1,15,16,17,18]. Moreover, these antimicrobial peptides are a viable strategy to replace conventional antibiotics or to potentiate their effects against pathogens, holding great promise (both purified and partially purified) in the control of harmful target bacteria.
Some Staphylococcus species, especially non-aureus staphylococci, are frequently found as commensals of humans and animals and are uncommonly associated with infections. Very recently, several coagulase-negative staphylococcal species have been included in a new genus, named Mammaliicoccus, which gathers, among others, the species M. sciuri (formerly Staphylococcus sciuri) [19]. The possible interest in bacteriocin-producing (BP) staphylococci and mammaliicocci (S/M) isolates or their antimicrobial compounds as antimicrobial, antitubercular, antifungal, antiviral, and/or anticancer strategies has been recently reported [20,21,22,23,24,25].
In this respect, a large collection of coagulase-positive and -negative staphylococcal strains (which include species now considered as mammaliicocci) was evaluated to detect and characterize commensal strains with antimicrobial activity. Based on the good results obtained in previous studies in terms of activity [26], added to the detection of bacteriocins at the genetic and protein level [27,28], we decided to select a collection of 15 BP-S/M strains with activity against G-positive bacteria (including different species of Enterococcus, Staphylococcus, and Listeria). Thus, in the present study, we aimed to further analyze the inhibitory capacities of the 15 selected BP-S/M strains (initially all of them identified as staphylococci) and their pre-purified bacteriocins against a wide collection of microorganisms used as indicators for agro-food and public health interest.

2. Results

2.1. Inhibitory Capacity of the BP-S/M Strains

Regarding the antimicrobial and antifungal activities of the 15 BP-S/M strains, Table 1 shows that all the 22 G-positive bacteria and the two molds were inhibited by at least one of the BP-S/M strains in one of the three conditions evaluated. Nevertheless, none of the BP-S/M strains showed antimicrobial activity (AA) against the three G-negative bacteria used as indicators in any of the assays evaluated. An example of the antimicrobial activity detected in this study by the three methods [spot-on-lawn, cell-free supernatants (CFS), and butanol (BT) extracts] is illustrated in Supplementary Figure S1.

2.1.1. Antimicrobial Activity of BP-S/M Strains Against the Indicators

The BP-S/M strains showed interesting antimicrobial activities against the 27 indicator microorganisms. The percentages of the BP-S/M strains (out of the 15 tested) that showed AA against each indicator microorganism are presented in Table 1, differentiated by the three different methods and the main application fields. Those indicators that were inhibited by ≥60% of the BP-S/M strains are colored in dark grey. Regarding the spot-on-lawn method, seven indicator strains were the most inhibited: methicillin-resistant S. aureus (MRSA) C5313, C1570 and C1532 strains; Listeria monocytogenes CECT911; Lactococcus garviae Om-Pe-HK-61; Pediococcus acidilactici A101; and Leuconostoc mesenteroides A103. Considering the activity of CFS, the inhibition rates of each indicator, when positive, ranged from 27% to 47% (with the highest rate observed in the case of L. monocytogenes). Moreover, none of the CFS of the 15 BP-S/M strains exhibited activity against the C. perfringens X9740, L. mesenteroides A103, or MRSA C5313 and C1532 strains. Finally, the inhibitory capacity of the BT extracts against the G-positive strains included in this study was notably higher than that observed with the other methods. It is worth noting the high percentages of inhibition (up to 93%) detected in the BT extracts against some indicator bacteria classified into the dairy livestock mastitis, swine zoonoses, and food safety fields of application. Nevertheless, both the CFS and BT extracts revealed limited inhibitory capacities (percentages from 13% to 33%) against lactic acid bacteria included in the aquaculture and wine-making fields. Finally, the antifungal activities of the BT extracts against pathogens involved in mushroom cultivation while preventing both the spore germination and mycelium growth of Trichoderma atroviride TAV1 (27% of BP staphylococci) and Cladobotryum mycophilum CM13900 (40%) are highlighted (Table 1).

2.1.2. Spectrum of Antimicrobial Activity of BP-S/M Strains Against Indicators

The spectrum of the antimicrobial activity of the 15 BP-S/M strains (grouped by the type of bacteriocin they produce) against the microorganisms used as indicators considering three methods: spot-on-lawn (n = 25), cell-free supernatant (CFS) (n = 25), and butanol extraction (BT) (n = 27), is shown in Figure 1. High percentages of indicator microorganisms were inhibited by the BP-S/M strains when the spot-on-lawn method (up to 84%), BT extraction (up to 89%), and CFS extracts (until 72%) were used. Among all of them, the MP1 producers showed the widest spectrum of activity by the three methods considered in this study. In relation to the BacSp222 producers, two profiles of antimicrobial activity were observed (Figure 1): (i) the S. pseudintermedius C8478 and C8479 strains revealed very similar antimicrobial activity patterns by the three methods tested; (ii) S. pseudintermedius C8189 showed lower activity with the BT or CFS extracts than that of the other BacSp222 producers. The S. chromogenes C9838 strain, carrying a gene of a circular bacteriocin, showed a higher percentage of inhibition against the indicators using the BT extract in comparison with that of the spot-on-lawn assay. Finally, the lanthipeptide-producing strains revealed different patterns of activities by the spot-on-lawn assays or by testing their BT extracts (Figure 1).
Supplementary Figure S2 represents the spectrum of the antimicrobial activity of the 15 BP-S/M strains (A: spot-on-lawn) and their bacteriocins (B: BT extracts) against the indicator microorganisms grouped by the fields of potential application. It is interesting to highlight that the Micrococcin P1 (MP1)-producing S/M strains were active against almost all the indicator categories. Moreover, the spot-on-lawn assays clearly showed more activity against the avian-related indicators than the BT extracts. Conversely, the BT extracts seemed to have more activity against the swine and food-related indicators (Supplementary Figure S2).

2.2. Intensity of the Inhibitory Action of the BP-S/M Strains or Their Bacteriocin Extracts Against the Indicator Microorganisms

The intensity of inhibition was calculated considering the inhibitory haloes of the 15 BP-S/M strains (spot-on-lawn) or their pre-purified extracts (BT) obtained against the indicator strains grouped into seven categories (related to the main fields of interest for potential application) and is represented in Figure 2. In this respect, the BT extracts showed special activity (with high intensity) against the indicators of interest for dairy livestock mastitis, aquaculture, and wine making (Figure 2B). Interestingly, the BT extracts of eight BP-S/M strains were active against one or two major mushroom pathogens, and, concretely, the MP1 producers (C5802, C5835, X3011, X3041) and lanthipeptide-like producers (C8609 and C9585) inhibited the growth in the cohabitant-symbiotic microbiota of the Cladobotryum and Trichoderma mycelia, respectively.
Moreover, the activity of the MP1 producers should be highlighted, which inhibited at least one indicator of each of the categories evaluated and revealed the strongest antimicrobial activity both by spot-on-lawn assays and BT extracts (Figure 2), especially the S. aureus C5802 and S. hominis C5835 strains. Conversely, the S. pseudintermedius C8189, S. aureus X3410, and S. epidermidis X3009 strains showed lower inhibitory capacities, acting only against one to four indicator categories, and also with reduced halo sizes.

2.3. Cross-Immunity and Potential Combinations of BP Strains

In order to evaluate the relatedness between the bacteriocins of the 15 BP-S/M strains, a cross-immunity assay was performed using all these BP strains both as bacteriocin producers and indicator bacteria (Figure 3). The white box indicates the absence of inhibitory activity in the pair producer–indicator bacteria, the grey box represents the existence of antimicrobial activity, and the black box indicates the absence of inhibitory activity between two BP-S/M strains both used as producers and indicator bacteria. As expected, the MP1 and BacSp222 producers did not exhibit inhibitory activity against strains of the same BP group due to the production of identical bacteriocins (boxes marked in red). The four MP1-producing strains inhibited almost all the BP-S/M strains (except other MP1 producers). Interestingly, all the MP1-producing strains were immune to the inhibitory activity of C8609, C6770, and X2969 as BP strains (carriers of genes encoding lanthipeptides) (Figure 3). Noteworthy, the MP1-producing S. hominis C5835 was immune to the inhibitory activity of 13 (including itself) of the BP-S/M strains (except for X3009 and C9832, carriers of genes of lanthipeptides), and what is more, it did not show inhibitory activity when testing against S. warneri X2969. Moreover, the three S. pseudintermedius producers of the BacSp222 bacteriocin were immune to the inhibitory activity of S. aureus C8609 (lanthipeptide-like-producer strain).
In this respect, considering the mutual interactions among the BP-S/M strains, the following potential BP-S/M combinations can be proposed: (i) two lanthipeptide-producing bacteria, such as C6770 + C8609, C6770 + C9585, C8609 + C9585, or X3410 + X2969; (ii) the lanthipeptide-producing S. warneri X2969 with the MP1-producing S. hominis C5835. In all these combinations, the two BP-S/M strains did not show inhibitory activity against the partner (marked in the black box in Figure 3).

2.4. Preliminary Characterization of Pathogenesis

A total of six out of the 15 BP-S/M strains were β-hemolytic, and all were coagulase-positive: three S. aureus (C8609, C6770, C5802) and three S. pseudintermedius (C8189, C8478, C8479). All the BP-producing coagulase-negative were negative for β-hemolysis. Moreover, five strains revealed gelatinase activity, including both coagulase-positive (three S. pseudintermedius: C8189, C8478, C8479) and coagulase-negative strains (S. hyicus C8581 and S. chromogenes C9838). Therefore, the subsequent screening of the safety and security of BP-S/M strains should be performed in the future to ensure their possible uses as protective cultures.

3. Discussion

Currently, the use of antimicrobial products with an environmental and respectful background is demanded. Bacteriocins have enormous research and potential application and hold great promise for the control of bacterial diseases, being an attractive alternative to antibiotics or chemical preservatives, and can also be used as semi-purified or bioactive compounds [29]. Several studies have indicated the health and economic benefits of bacteriocins or BP-S/M strains as protective cultures and as preventive treatments against diseases or in combination with other antimicrobial compounds for livestock production [1,30,31,32,33,34,35,36,37,38]. However, their application is still limited, at least regarding staphylococci and mammaliicocci.
The discovery and characterization of new antimicrobials, such as S/M bacteriocins, will likely become an important step in the fight against antimicrobial resistance. In this respect, the present study aims to contribute to the knowledge of the potential applications of staphylococcins (including those of mammaliicocci; in this case, we propose to use the term mammaliicoccin) and the BP-S/M strains. This study shows the interesting antimicrobial activities of BP-S/M strains verified by several methods against harmful microorganisms, including MDR bacteria, involved in relevant infections and important problems of agro-food application fields such as dairy livestock mastitis, poultry pathogen zoonoses, swine zoonoses, food safety, aquaculture, mushroom cultivation, and wine making. Overall, each G-positive bacteria tested as an indicator was inhibited by the BT extract of at least two BP-S/M strains, whereas none of the producers revealed antimicrobial activity against the G-negative indicators. These results are consistent with the available literature, wherein it is assumed that commensal bacteriocin-producing strains inhibit specific colonizing pathogens [39]. Concretely, staphylococcins often show antimicrobial activity against related bacteria, and normally G-positive bacteria [40].
As expected, wide inhibitory activities were detected when BT extracts were used. It is worth noting that 10 BP-S/M strains had antimicrobial activity against more than 40% of the indicators, regardless of their assigned category (S. aureus C5802; S. hominis C5835; M. sciuri X3041 and X3011; S. pseudintermedius C8478 and C8479; S. aureus C8609 and C6770; S. warneri X3009; and S. hyicus C9585). These BP strains produced Micrococcin P1, BacSp222, and lanthipeptide-like bacteriocins, which have previously been reported to inhibit critical G-positive harmful bacteria such as Mycobacterium tuberculosis, MRSA, Listeria monocytogenes, vancomycin-resistant enterococci, and Clostridium difficile, among others [16,41,42,43].
Taking into account the results obtained in relation to the harmful bacteria of interest in livestock production, we highlight that all the BP-S/M strains evaluated in this study inhibited at least one indicator strain included in the bovine mastitis and swine zoonoses categories, both considered of great relevance in livestock production. Moreover, the assays performed in this study revealed that the strains included in the swine pathogen category were the most susceptible to BT extracts, while both BP-S/M strains (spot-on-lawn) and their bacteriocins (BT extracts) could be active against strains included in the dairy mastitis pathogen cathegory. In this respect, the use of bacteriocins as natural antimicrobial substances for mastitis control and prevention has already been considered [38,44,45]. A recent ex vivo study proposes the use of staphylococcins against pathogens isolated from bovine mastitis, evidencing that, after 24 h of incubation, aureocin A53 caused a strong reduction in staphylococcal or streptococcal populations, eliminating all the detectable viable cells [46]. In addition, it is of great interest to solve the problem of the community transmission of S. aureus, and especially MRSA-CC398, between pigs and humans, a livestock-associated lineage considered an emerging public health problem. On this basis, a recent ex vivo and in vivo murine model study confirmed the protective role of the porcine skin microbiota, including bacteria-producing antimicrobial substances, against MRSA in a colonization model [47]. Here, cross-immunity assays allowed us to detect five bacterial combinations of lanthipeptide-producing strains and the MP1-producing S. hominis C5835 strain of interest for future studies by enhancing their antimicrobial activity against MRSA, among other pathogens. Moreover, in a previous study [28], the MP1-producing C5835 strain and its pre-purified extract revealed an intense inhibitory capacity against an MRSA-MDR-CC398 strain, which allows us to consider this producing strain as a good candidate for potential applications. Similar results have recently been presented, suggesting the use of BP strains with synergistic interactions in vivo as consortium probiotic supplements to promote the health of piglets [48].
Regarding poultry, it is important to highlight the potential use of BP strains as suitable alternatives to antibiotics to combat or prevent infections of relevant pathogens in poultry farming, such as C. perfringens and MRSA, as well as for improving the performance and productivity of birds [49,50]. In this respect, the results presented in this study revealed BP-S/M strains with potent inhibitory activity by different methods against indicators included in the avian pathogen zoonoses category, such as C. perfringens, MRSA, and E. cecorum. Concretely, it is worth noting that the four MP1-producing strains (C5802, C5835, X3041, and X3011) and three lanthipeptide-like-producing (C8609, C6770, and X3410) S/M strains inhibited all three indicators of the avian pathogen category tested by the spot-on-lawn assay.
The aquaculture system field was also considered in this study and one Lactococcus garviae strain was included as an indicator, as it is regarded as an emerging zoonotic pathogen [51]. Focusing on our results, the Lactococcus garviae strain was inhibited by 60% of the BP staphylococci (spot-on-lawn), as well as by their extracts. The potential use of BP-S/M strains and their bacteriocins in this sector could therefore be proposed for further studies.
Regarding the food industry, foodborne disease causes an estimated 48 million illnesses and 3000 deaths annually, with important economic costs [52]. Among the most harmful microorganisms, the negative effects of Salmonella, L. monocytogenes, S. aureus, C. perfringens, and E. coli contamination can be highlighted [10]. In this study, several extracts demonstrated interesting inhibitory effects against the G-positive strains included in the food pathogen category. Attending to the food industry, nisin is undoubtedly the most widely used and effective bacteriocin against foodborne pathogens, although the use of other bacteriocins has gained importance due to their wide range of applications in the food industry [53]. In this respect, a previous study carried out by our group compared the antimicrobial activity of non-purified extracts of MP1-producing strains with that of a non-pure nisin extract (2.5%, equivalent to 5 µg/mL) and the relevant antimicrobial activities of MP1 were detected against S. aureus MRSA and L. monocytogenes [28].
Moreover, lactic acid bacteria (LAB) have been widely used in the food industry and concretely represent a key factor for wine production responsible for malolactic fermentation [54]. In this respect, synergism between commercial bacteriocins and preservatives such as sulfur dioxide has been reported in controlling the growth of LAB in the different steps of wine production: Oenococcus, Lactobacillus, Leuconostoc, and Pediococcus [55]. In this study, we detected BP-S/M strains and some of their BT extracts with interesting activities against LAB included in the wine-making category, revealing their potential interest in this field of application.
With respect to mushroom cultivation, fungal diseases are among the most serious disorders damaging and affecting the quality of the yields of relevant crops such as mushroom [56,57]. However, the scarce range of available products in addition to the occurrence of resistance derived from the continuous exposure to the same active substances evidence the need for alternative treatments [58]. Concretely, we focus on two of the most harmful fungal diseases affecting cultivated mushrooms: cobweb disease (Cladobotryum spp.) and green mold disease (Thrichoderma spp.). Interestingly, antifungal activity was observed in six out the 15 BP-S/M strains tested in this study towards these mycoparasites, also inhibiting the mycelial growth of Cladobotryum mycophilum by the noted antagonism towards its respective cohabitant-symbiotic microbiota, suggesting that the growth of this harmful fungi could be repressed by the observed detrimental effect on its mutualistic microbiota. Thus, bacteriocins could be of interest as a biological control agent of some mycoparasites by means of their detrimental action on essential bacteria conforming to the pathogen microbiome. Nevertheless, more research should be performed in this field to deepen the understanding of these activities and the potential mechanisms of action. This is a singular observation in our approach since, commonly, mycoparasites are commercially treated by chemical fungicides, but the scarcely known fungal–bacterial interactions could play a role in facilitating pathogen control through active bacteriocin producers. This configuration may have a direct impact on the yield performance and disease occurrence [59,60], contributing to enhancing the production of protein alternatives to meat-based, Western diets.
Overall, the present study demonstrates that the use of both BP-S/M strains and their bacteriocins as pre-purified BT extracts could be considered in further applications. Specifically, the wide spectrum and intense inhibitory activity of the MP1 producers against all the indicator categories and by all the conditions evaluated are remarkable.
Micrococcin P1 is a 26-membered ring thiopeptide isolated from a wide variety of sources ranging from soil to cheese. MP1 has been reported to primarily inhibit G-positive bacteria, although it has also been described as a good antitubercular compound [43]. Regarding its mode of action, MP1 is able to inhibit bacterial protein synthesis, especially the elongation step, by binding to the GTPase-associated region of the ribosome involving the protein L11 and related rRNA complex [43,61]. Additionally, it has been reported that mutations in ribosomal protein L11 in mycobacteria is associated with resistance to MP1 [43].
Finally, it is important to consider the limitations of this study regarding the limited number of indicators in each category and the criteria used to establish the respective groups. Certainly, the inhibitory activities observed in this study depend on the established categories, on the producing strains, and on the type of bacteriocin; however, the results elucidate promising alternatives to antibiotics in the agro-food and public health sectors.

4. Materials and Methods

4.1. Bacteriocin-Producing and Indicator Strains

The 15 BP-S/M strains included in this work were obtained and well characterized as bacteriocin producers at the genetic and protein level in previous studies [26,27,28]. A summary of their molecular characterization after whole-genome analysis and their contents of bacteriocins is shown in Supplementary Table S1, and a briefly summarization is shown in Table 2. These BP-S/M strains contained the bacteriocin gene clusters encoding the MP1 thiopeptide (S. aureus C5802, Staphylococcus hominis C5835, and Mammaliicoccus sciuri X3041 and X3011); lanthipeptides (S. aureus C8609, S. aureus X3410, S. aureus C6770, Staphylococcus epidermidis X3009, Staphylococcus warneri X2969, Staphylococcus simulans C9832, and Staphylococcus hyicus C9585); circular bacteriocins (Staphylococcus chromogenes C9838); and the BacSp222 (S. pseudintermedius C8189, C8478, and C8479).
The inhibitory activity of these 15 strains was evaluated against selected harmful strains used as indicators (22 G-positive strains, three G-negative strains, and two molds), including multidrug-resistant (MDR) and zoonotic strains of relevance. Indicator strains were selected based on their species and/or antimicrobial resistance phenotypes and were classified into seven categories based on their main fields of potential application (Table 1): dairy livestock mastitis, avian pathogen zoonoses, swine zoonoses, food safety, aquaculture, wine making, and mushroom cultivation. Although, for simplicity, each of these indicator strains was categorized into only one potential application field, it is important to remark that they could be included for the study of several. In addition to most of the indicator microorganisms were G-positive bacteria, and three G-negative bacteria (Escherichia coli, Salmonella, and Pseudomonas aeruginosa) were also included as indicators and were considered separately (outside the potential application fields). The antimicrobial activity of these BP-S/M strains against six indicator bacteria of the genera Staphylococcus, Enterococcus, and Listeria has already been tested in previous studies [28].
Moreover, Cladobotryum mycophilum CM13900 and Trichoderma atroviride TAV1, isolated from commercial mushroom farms in La Rioja (Spain), were used as indicators to study the possible antifungal activity (AF) of the BP-S/M strains. These mycopathogens are responsible for cobweb and green mold on mushroom farms.

4.2. Culture Media and Growth Conditions

Brain Heart Infusion (BHI) (Condalab, Madrid, Spain) was used as the standard growth medium for both liquid and solid cultures (1.5% of agar). Antimicrobial activity determinations were performed by the “spot-on-lawn” assay in plates containing one layer of 10 mL of tryptic soy agar (TSA) supplemented with 0.3% of yeast extract (Condalab Spain) and a second layer of 5 mL of tryptic soy broth (TSB) supplemented with 0.3% yeast extract (Condalab, Madrid, Spain) and 0.7% agar tempered at 45 °C.
Anaerobic conditions were used for the Clostridium perfringens, Pediococcus spp., Lactobacillus plantarum, and Leuconostoc mesenteroides indicators. Columbia agar with 5% sheep blood (BioMérieux, France) was used when the indicator strain was Streptococcus suis and potato dextrose agar media (PDA) was used in the case of molds. The 15 BP-S/M strains and the indicator microorganisms were grown in BHI agar incubated overnight at 37 °C during 24 h, except those indicators selected as representatives for mushroom cultivation and aquaculture applications, which were incubated at 25 °C during 24 h to 96 h in the case of molds and 48 h for lactic acid bacteria.

4.3. Detection of Antimicrobial Activity (AA)

The AA of the BP-S/M strains was evaluated by the spot-on-lawn method (n = 25 indicators), and the inhibitory capacity of two different bacterial extracts: CFS (n = 25) and BT (n = 27 indicators) was tested by agar diffusion assays. The two molds were only tested as indicators with the BT extracts.
For the spot-on-lawn method, indicator strains were resuspended in Brain Heart Infusion (BHI) broth to obtain a 0.5 McFarland (108 CFU). Later, 10 μL of these cultures were added to tubes containing 5 mL of semisolid TSA (tryptic soy broth (TSB) containing 0.5% yeast extract and 0.7% agar), and the mixtures were poured onto TSA agar plates with 0.3% yeast extract. After that, the 15 BP-S/M strains were spotted on the surface of each plate (previously seeded with each indicator strain) using sterile toothpicks, and plates were incubated overnight at 37 °C and analyzed for AA. All determinations were performed in duplicate.
Regarding agar diffusion assays, CFS and BT extracts were obtained from the 15 BP-S/M strains. For preparation of CFS, strains were grown in BHI broth medium for 24 h at 37 °C under continuous shaking, centrifuged (4500 rpm, 10 min), and sterilized by filtration through a low-protein-binding 0.45 µm Millipore filter. BT extracts were obtained by incubating boiling sterilized CFS (100 °C, 5 min) with 1-butanol at a ratio of 1:3 and were shacked for 1 h at 37 °C. Samples were centrifuged at 4500 rpm for 15 min, the aqueous phase was discarded after centrifugation, and the organic phase was evaporated at 44 °C under reduced pressure. The BT extracts were resuspended in the minimum needed volume of dimethyl sulfoxide (DMSO) (0.7–0.9 mL). Indicator strains were resuspended in 0.9% sterile saline solution at a bacterial concentration of 0.5 McFarland turbidity and were seeded on TSA plates. Wells of 6 mm diameter were punched into the inoculated agar plates and filled with 50 µL of the CFS samples, whereas 10 µL of the BT extract was added to a blank disc. Plates were incubated overnight at the corresponding temperatures, and diameters of inhibition zones were measured (mm) and recorded. All determinations were repeated.

4.4. Detection of Antifungal Activity (AF)

The mold spores were collected in sterile water and rubbed onto the PDA surface with a glass rod (100 µL per plate). The spore suspensions were quantified by using a counting chamber, adjusted to 104 spores/mL, and used as inoculum. The disc diffusion assay was used to evaluate the AF. Thus, 10 µL of BT extracts of the 15 BP-S/M strains was added to sterile discs. Dry discs were placed in PDA plates inoculated with mold spore suspension.

4.5. Intensity of the Inhibitory Capacity

The inhibition intensity of the 15 BP-S/M strains against the 27 indicator strains was determined considering the inhibition diameter by measuring the haloes in millimeters (mm) after spot-on-lawn and BT assays, as follows: area of zone πr2−area of well πr2 [62].

4.6. Cross-Immunity Among the BP Staphylococci

The cross-immunity assay was performed to determine the relatedness of the bacteriocins produced in the selected collection of BP-S/M strains (although it belongs to the same class of antimicrobial peptides). Moreover, the cross-immunity assay allows us to propose bacterial combinations to enhance their own activities against other bacteria. Thus, the 15 BP-S/M strains included in this study were evaluated to explore their antimicrobial activity by the spot-on-lawn method described above using each BP-S/M strain both as a producer and target indicator strain [62].

4.7. Hemolysis and Gelatinase Activities

Overnight cultures of BP-S/M strains in BHI broth were spotted onto Columbia agar with 5% sheep blood (BioMérieux, France) and then incubated at 37 °C overnight. Hemolytic activities of the strains were recorded by the presence of beta (β) hemolysis when detecting a clear and complete hemolysis of blood cells around the BP-inoculated strain.
Moreover, gelatinase activity was evaluated in BP-S/M strains, which were spotted onto TSA plates containing 1.5% of skim milk and incubated overnight at 37 °C. Then, plates were cooled to room temperature for 2 h and evaluated for the appearance of a transparent halo around the colonies (positive for gelatinase test) [49].

5. Conclusions

The present study shows the promising antimicrobial capacity of both BP-S/M strains and their bacteriocins (pre-purified BT extracts) against harmful microorganisms classified into different categories within the agro-food and public health fields of application. In addition, cross-immunity assays helped us to propose five potential combinations of BP-S/M strains to enhance their activity against specific pathogens. Finally, the BP-coagulase-negative and mammaliicocci strains lacked beta-hemolytic and gelatinase activities, so further studies should be performed to deepen the applicability of the BP-S/M strains and their bacteriocins as protective cultures and antimicrobial agents, respectively. In addition to all the above, the three coagulase-negative producers of the MP1 bacteriocin, and especially the S. hominis C5835 strain, and their staphylococcins have shown themselves to be good candidates for agro-food applications due to their wide spectrum and high intensity of inhibition, especially against indicators included in the dairy livestock mastitis, avian pathogen zoonosis, aquaculture, and wine-making categories, alone or in combination with other BP staphylococci (S. warneri X2969).

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/antibiotics14010097/s1, Table S1: Characteristics of the 15 bacteriocin-producing (BP) staphylococci and mammaliicocci (S/M) strains included in this study. Figure S1: Antimicrobial activity of five bacteriocin-producing staphylococci and mammaliicocci strains against different indicator bacteria represented from left to right (1–3): 1. bacteria (spot-on-lawn), 2. cell-free supernatant (diffusion assay), and 3. butanol extracts. (A) Staphylococcus aureus C5802 against MRSA X3963; (B) Staphylococcus hominis C5835 against Enterococcus faecalis C9951; (C) Mammaliicoccus sciuri X3011 against Listeria monocytogenes; (D) Staphylococcus aureus C8609 against Streptococcus gallolyticus; (E) Mammaliicoccus sciuri X3041 against Bacillus cereus. Figure S2: Spectrum of antimicrobial activity of the 15 bacteriocin-producing (BP) staphylococci/mammaliicocci (S/M) strains against the collection of microorganisms used as indicators considering the seven categories of application for which they were selected (marked in different colors). The 15 BP-S/M strains were grouped according to the type of bacteriocin produced. The figure represents the percentage of indicator strains inhibited by each of the BP staphylococci organized by the two methods (number of indicators tested): (A): spot-on-lawn (n = 22); (B): butanol extracts (BT, n = 24).

Author Contributions

R.F.-F., C.T. and C.L. designed and contextualized the study; R.F.-F., performed most of the experiments; A.C.-B., C.G.-A., T.Á.-G. and R.F.-P. contributed to the experimental laboratory work related to the butanol extracts and the growth indicator microorganisms; R.P. and J.C. contributed to the experiments with molds; R.F.-F., C.L., J.C., M.Z. and C.T. analyzed the data; R.F.-F., C.T., C.L. and M.Z. prepared the first draft of the manuscript; C.T. and C.L. supervised the study; C.T. and M.Z. contributed to the project funding. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by MICIU/AEI/10.13039/501100011033 and FEDER, UE (project PID2022-139591OB-I00). R.F.-F. had a predoctoral contract (FPU18/05438) during the experimental work of this study. J.C. is the recipient of a Ramón y Cajal contract [RYC2021-032796-I], funded by MCIN/AEI/10.13039/501100011033 (Ministerio de Ciencia e Innovación, Spain) and the European Union “Next Generation EU”/PRTR.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data generated and reported during this study are included or indicated in the published article.

Conflicts of Interest

The authors declare no competing interests.

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Antibiotics 14 00097 g001
Figure 1. Spectrum of antimicrobial activity of the 15 bacteriocin-producing (BP) staphylococci and mammaliicocci (S/M) strains against the microorganisms used as indicators (number) considering three methods: spot-on-lawn (n = 25), cell-free supernatant (CFS) (n = 25), and butanol extraction (BT) (n = 27). The 15 BP-S/M strains were grouped according to the type of bacteriocin produced. The percentage of inhibition represents the percentage of the indicator microorganisms inhibited by each of the 15 BP-S/M strains. The antifungal activity of the 15 BP-S/M strains against the two molds included in this study was only evaluated with the BT extracts by disc diffusion assay.
Figure 1. Spectrum of antimicrobial activity of the 15 bacteriocin-producing (BP) staphylococci and mammaliicocci (S/M) strains against the microorganisms used as indicators (number) considering three methods: spot-on-lawn (n = 25), cell-free supernatant (CFS) (n = 25), and butanol extraction (BT) (n = 27). The 15 BP-S/M strains were grouped according to the type of bacteriocin produced. The percentage of inhibition represents the percentage of the indicator microorganisms inhibited by each of the 15 BP-S/M strains. The antifungal activity of the 15 BP-S/M strains against the two molds included in this study was only evaluated with the BT extracts by disc diffusion assay.
Antibiotics 14 00097 g001
Antibiotics 14 00097 g002
Figure 2. Intensity of inhibition of the 15 BP staphylococci and mammaliicocci (S/M) strains against the indicator microorganisms classified into seven categories based on their potential applications and represented in colors: two methods were used (numbers of indicators): (A) spot-on-lawn (n = 22), (B) BT extracts (n = 24). The intensity of the inhibitory activity was calculated by the inhibition zones as indicated in the Materials and Methods (in mm2): white (0); light grey (1–49); dark grey (50–150); rose (>150).
Figure 2. Intensity of inhibition of the 15 BP staphylococci and mammaliicocci (S/M) strains against the indicator microorganisms classified into seven categories based on their potential applications and represented in colors: two methods were used (numbers of indicators): (A) spot-on-lawn (n = 22), (B) BT extracts (n = 24). The intensity of the inhibitory activity was calculated by the inhibition zones as indicated in the Materials and Methods (in mm2): white (0); light grey (1–49); dark grey (50–150); rose (>150).
Antibiotics 14 00097 g002
Antibiotics 14 00097 g003
Figure 3. Cross-immunity assay between the 15 bacteriocin producing (BP) staphylococci/mammaliicocci (S/M) strains classified according to the bacteriocins produced. All BP-S/M strains were used both as producers (in columns) and as indicators (in lines) in this assay. The BP-S/M strains that lacked antimicrobial activity when they were used both as indicator or producer bacteria are marked with a red box, for cases in which they carried exactly the same bacteriocin (MP1 and BacSp222).
Figure 3. Cross-immunity assay between the 15 bacteriocin producing (BP) staphylococci/mammaliicocci (S/M) strains classified according to the bacteriocins produced. All BP-S/M strains were used both as producers (in columns) and as indicators (in lines) in this assay. The BP-S/M strains that lacked antimicrobial activity when they were used both as indicator or producer bacteria are marked with a red box, for cases in which they carried exactly the same bacteriocin (MP1 and BacSp222).
Antibiotics 14 00097 g003
Table 1. Percentages of the 15 bacteriocin-producing (BP) staphylococci and mammaliicocci (S/M) strains with inhibitory activity against each of the 27 indicator strains classified into eight categories, using three methods (spot-on-lawn; cell-free supernatant, CFS; butanol extracts, BT).
Table 1. Percentages of the 15 bacteriocin-producing (BP) staphylococci and mammaliicocci (S/M) strains with inhibitory activity against each of the 27 indicator strains classified into eight categories, using three methods (spot-on-lawn; cell-free supernatant, CFS; butanol extracts, BT).
Main Field of Interest Indicator Strains bPercentage of BP Strains with Inhibitory Activity Against the Indicator a
Spot-on-lawnCFSBT
Dairy livestock mastitisStreptococcus agalactie X9738274087
Streptococcus dysgalactie X9739332787
Streptococcus gallolyticus X9742403360
Staphylococcus aureus C7246 MRSA b332733
S. aureus X3548 MRSA b532760
Enterococcus faecium C2321 VanR b532760
Avian pathogen zoonosesClostridium perfringens X974053027
Enterococcus cecorum X3809473387
S. aureus C5313 MRSA b60047
Swine zoonosesStreptococcus suis X2060472780
S. aureus C1570 MRSA-CC398 b672773
S. aureus X3963 MRSA b533393
Enterococcus faecalis C9951 LZD-R b332767
Food safetyS. aureus C1532 MRSA b60073
Bacillus cereus X10062472787
Listeria monocytogenes CECT911734773
AquacultureLactococcus garviae Om-Pe-HK-61602713
Carnobacterium maltaromaticum St-PS-HK-63402713
Wine makingPediococcus pentosaceus A100472713
Pediococcus acidilactici A101602713
Lactobacillus plantarum A10203320
Leuconostoc mesenteroides A10367020
Mushroom cultivationTrichoderma atroviride TAV1NTNT27
Cladobotryum mycophilum CM13900NTNT40
Gram-negative cSalmonella spp. X10061000
Escherichia coli ATCC25922000
Pseudomonas aeruginosa PAO1000
a The indicator strains inhibited by ≥60% of the BP-S/M strains are marked with the dark-grey color, those inhibited by 20% to 60% of the BP-S/M strains are marked in the light-grey color, and those inhibited by less than 20% of the BP-S/M strains are marked in the white color. b For some indicator strains, specific characteristics are included: MRSA: methicillin-resistant Staphylococcus aureus; VanR: vancomycin-resistant; LZD-R: linezolid-resistant; CC: clonal complex. c Gram-negative bacteria were considered separately due to the fact they were not inhibited by any of the BP-S/M strains included in this study or by their pre-purified extracts. Abbreviations: CECT, Spanish Type Culture Collection; ATCC, American Type Culture Collection; NT, not tested.
Table 2. Collection of the 15 bacteriocin-producing (BP) strains included in this study a.
Table 2. Collection of the 15 bacteriocin-producing (BP) strains included in this study a.
ID CodeSpecieOriginBacteriocin Detected
C5802S. aureusEnvironment–waterMicrococcin P1
C5835S. hominisEnvironment–waterMicrococcin P1
X3011S. sciuri bFood–chickenMicrococcin P1
X3041S. sciuri bFood–chickenMicrococcin P1
C8189S. pseudintermediusHuman–dogBacSp222
C8478S. pseudintermediusHuman–dogBacSp222
C8479S. pseudintermediusHuman–dogBacSp222
C9838S. chromogenesWild animal–mammalCircular
C8609S. aureusWild animal–mammalLanthipeptide
X3410S. aureusFood–chickenLanthipeptide
C6770S. aureusWild animal–mammalLanthipeptide
X2969S. warneriFood–chickenLanthipeptide
X3009S. epidermidisFood–chickenLanthipeptide
C9832S. simulansWild animal–mammalLanthipeptide
C9585S. hyicusWild animal–mammalLanthipeptide
a Data on the strains were obtained in previous studies [27,28]. b S. sciuri is now included in the new Mammaliicoccus genus and is considered M. sciuri.
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Fernández-Fernández, R.; Lozano, C.; Campaña-Burguet, A.; González-Azcona, C.; Álvarez-Gómez, T.; Fernández-Pérez, R.; Peña, R.; Zarazaga, M.; Carrasco, J.; Torres, C. Bacteriocin-Producing Staphylococci and Mammaliicocci Strains for Agro-Food and Public Health Applications with Relevance of Micrococcin P1. Antibiotics 2025, 14, 97. https://doi.org/10.3390/antibiotics14010097

AMA Style

Fernández-Fernández R, Lozano C, Campaña-Burguet A, González-Azcona C, Álvarez-Gómez T, Fernández-Pérez R, Peña R, Zarazaga M, Carrasco J, Torres C. Bacteriocin-Producing Staphylococci and Mammaliicocci Strains for Agro-Food and Public Health Applications with Relevance of Micrococcin P1. Antibiotics. 2025; 14(1):97. https://doi.org/10.3390/antibiotics14010097

Chicago/Turabian Style

Fernández-Fernández, Rosa, Carmen Lozano, Allelen Campaña-Burguet, Carmen González-Azcona, Tamara Álvarez-Gómez, Rocío Fernández-Pérez, Raquel Peña, Myriam Zarazaga, Jaime Carrasco, and Carmen Torres. 2025. "Bacteriocin-Producing Staphylococci and Mammaliicocci Strains for Agro-Food and Public Health Applications with Relevance of Micrococcin P1" Antibiotics 14, no. 1: 97. https://doi.org/10.3390/antibiotics14010097

APA Style

Fernández-Fernández, R., Lozano, C., Campaña-Burguet, A., González-Azcona, C., Álvarez-Gómez, T., Fernández-Pérez, R., Peña, R., Zarazaga, M., Carrasco, J., & Torres, C. (2025). Bacteriocin-Producing Staphylococci and Mammaliicocci Strains for Agro-Food and Public Health Applications with Relevance of Micrococcin P1. Antibiotics, 14(1), 97. https://doi.org/10.3390/antibiotics14010097

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