- Academic Editor
Background: Bacterial endophytic communities associated with medicinal plants synthesize a plethora of bioactive compounds with biological activities. Their easy isolation and growth procedures make bacterial endophytes an untapped source of novel drugs, which might help to face the problem of antimicrobial resistance. This study investigates the antagonistic potential of endophytic bacteria isolated from different compartments of the medicinal plant O. heracleoticum against human opportunistic pathogens. Methods: A panel of endophytes was employed in cross-streaking tests against multidrug-resistant human pathogens, followed by high-resolution chemical profiling using headspace-gas chromatography/mass spectrometry. Results: Endophytic bacteria exhibited the ability to antagonize the growth of opportunistic pathogens belonging to the Burkholderia cepacia complex (Bcc). The different inhibition patterns observed were related to their taxonomic attribution at the genus level; most active strains belong to the Gram-positive genera Bacillus, Arthrobacter, and Pseudarthrobacter. Bcc strains of clinical origin were more sensitive than environmental strains. Cross-streaking tests against other 36 human multidrug-resistant pathogens revealed the highest antimicrobial activity towards the Coagulase-negative staphylococci and Klebsiella pneumoniae strains. Interestingly, strains of human origin were the most inhibited, in both groups. Concerning the production of volatile organic compounds (VOCs), the strain Arthrobacter sp. OHL24 was the best producer of such compounds, while two Priestia strains were good ketones producers and so could be considered for further biotechnological applications. Conclusions: Overall, this study highlights the diverse antagonistic activities of O. heracleoticum-associated endophytes against both Bcc and multidrug-resistant (MDR) human pathogens. These findings hold important implications for investigating bacterial endophytes of medicinal plants as new sources of antimicrobial compounds.
The World Health Organization official definition of traditional medicine is “the sum total of the knowledge, skills, and practices based on the theories, beliefs, and experiences indigenous to different cultures, whether explicable or not, used in the maintenance of health, as well as in the prevention, diagnosis, improvement or treatment of physical and mental illnesses” [1]. Medicinal plants have been used since ancient times, and traditional medicine is still practiced in a great portion of the world’s population, especially in developing countries of Africa and Asia, where these practices are perceived as more accessible and affordable, compared to modern medicine [2]. However, it is important to acknowledge that within traditional medicine, certain practices, approaches, or substances have questionable efficacy or were scientifically debunked. Among the different approaches of traditional medicine, phytotherapy has garnered a more extensive body of scientific knowledge. The curative potential of medicinal plants can be attributed to the synthesis of a wide variety of phytochemicals, which have evolved over millions of years; such compounds are involved in the interaction among organisms and in the plant response to different biotic and abiotic stresses [3, 4]. Indeed, medicinal plants represent a valuable and manifold source of natural compounds with pharmaceutical potential: in many cases, the isolation and characterization of natural compounds have led to the development of widely used drugs or served as initiating steps in drug discovery [5, 6].
The emergence of antimicrobial resistance, caused by the extensive and/or inappropriate use of antibiotics, has resulted in making many currently available antimicrobial drugs ineffective. The advent and spread of Multi-Drug Resistant (MDR) bacterial pathogens have become a significant and urgent public health issue, thought to put at risk 10 million lives/year in the Southeast Asian region by the year 2050 [7]. Considering the rapid worldwide dissemination of MDR clinical isolates, the discovery of new antimicrobial agents is of paramount importance [8]. Various methods have been used to obtain new effective molecules, including synthetic and combinatorial chemistry, or molecular modeling, but despite the initial interest in such synthetic techniques, the focus has recently shifted towards the employment of medicinal plants [9].
A great number of officinal plants have been recognized as a resource of antimicrobial compounds potentially effective in the treatment of MDR bacterial infections [10]. Many plants secondary metabolites, such as tannins, alkaloids, phenolic compounds, steroids, and flavonoids exhibit a growth-inhibitory action, preventing biofilm production and inhibiting bacterial virulence in vitro [11]; furthermore, there is evidence for the improvement of the activity of conventional antibiotics when used in combination with plant-derived compounds [12]. Hence, medicinal plants offer a very promising source of novel antibiotics in the fight against MDR bacteria: they are more available and accessible in small local communities, cheaper to purchase, easier to administrate, and have better biodegradability as compared to other available antibiotics [11]. Their structural variety is enormous and influences the antimicrobial activity they exert against different pathogenic microbes [13]. Furthermore, the effectiveness of medicinal plant extracts and essential oils (EOs) appears to be related to the synergistic effect between the multiple bioactive compounds, which lowers the chances for MDR bacteria to become resistant [14].
However, there are some challenges related to the use of plant natural products as antimicrobial pharmaceuticals, such as the lack of standardization during the preparation and storage of plant materials [15], the variation in the composition of plant extracts and EOs, which is influenced by both abiotic and biotic factors [16], or the possibility that compounds that have shown antimicrobial activity in vitro may have little or no effect in vivo [17]. Additionally, the isolation and the characterization of single compounds with the desired biological activity can be time-consuming and, in most cases, require large amounts of plant material, raising some issues regarding biodiversity conservation, as some medicinal plants are endangered and/or endemic species [18].
The genus Origanum represents one of the most important groups of aromatic and medicinal plants of the Lamiaceae family, distributed in warm and mountainous areas. Oregano is generally used as a spice in cooking, but it has also been employed in traditional medicine to treat respiratory disorders, stomachache, and rheumatoid arthritis, as well as a diuretic, anti-urolithic, and antimicrobial agent [19]. To date, over 100 volatile and nonvolatile compounds have been identified in the EO and extracts of Origanum species, the most represented being carvacrol, thymol, and rosmarinic acid [19]. Origanum antimicrobial efficacy is mainly exerted by its EO, making it an ideal candidate in various fields of applications, such as food preservation, natural medicine, and agricultural pest management [20]. Indeed, the EO and/or its main constituents induced remarkable inhibitory effects against MDR pathogenic bacterial strains [21, 22, 23, 24]. However, when considering the broader Origanum health benefits, the synergy of various compounds, such as phenolic compounds, flavonoids, and other polyphenols, plays a crucial role [25]. The two volatile main compounds of Origanum EO, carvacrol and thymol, are “Generally Recognized as Safe” (ESO, GRAS-182.20) for human usage. However, the usage of those substances at the wrong concentration or for a prolonged time can induce toxic side effects to the liver, kidneys, and nervous system, and procure irritating effects to skin and mucous membranes [25].
It is widely recognized that plants’ health and metabolism can be strongly influenced by the presence of specific microbial endophytes. Endophytic bacteria and/or fungi were found in nearly all vascular plant species studied: they can occupy all organs of their plant host, and some of them are seed-borne [26]. The relationship between plants and bacteria has evolved ever since plants appeared on Earth, making it possible for microbes to come up with peculiar genetic systems and metabolic pathways [27]. Little is known about how the plant determines and modulates the endophytic bacterial community composition and structure, or how endophytes influence their host, but their interaction is thought to be flexible and dynamic [28].
Medicinal plant studies mainly focused on their synthesis of bioactive phytochemicals, but recently the interest started to shift towards the exploration of their bacterial endophytic communities, as they produce a notable number of bioactive compounds that share the same or similar anticancer, anti-inflammatory, antioxidant, and antimicrobial activities [27]. Medicinal plant-associated bacterial endophytes have the potential to produce bioactive metabolites and/or modulate the plant’s secondary metabolism; furthermore, some secondary metabolites may be the result of the combined metabolism of both the bacteria and their host [28, 29]. Bacterial endophytes can produce different classes of bioactive compounds with biological activities, such as alkaloids, polyketones, lactones, phenolic and organic acids, flavonoids, saponins, steroids, terpenoids, phenols, peptides, and polyketides, but still represent an almost untapped source of natural molecules [27, 28, 30, 31]. Obtaining such compounds from endophytes might offer numerous benefits: their easy isolation and growth procedures allow the preservation of the host plant, as minimal plant material is required, and the production of biologically active compounds can be increased through microbial fermentation [31]. Consequently, the investigation of bacterial endophytes associated with medicinal plants may provide a solid basis for the development of novel drugs and might help to face the antimicrobial resistance issue.
Considering their potential biotechnological and pharmaceutical applications, endophytic bacterial strains were isolated from the medicinal and aromatic plant Origanum heracleoticum L. [32], also known as O. vulgare ssp. viridulum or O. virens Hoffmanns. & Link. O. heracleoticum is an aromatic herb widespread in the Mediterranean area. The main component of its EO is carvacrol, which is thought to be involved in its antibacterial and antifungal activity [33, 34, 35]. To the best of our knowledge, the antimicrobial potential of O. heracleoticum-associated bacterial endophytes has not been investigated, except for seed-associated endophytes [36]. In this study, their antibacterial activity against MDR human pathogenic strains was tested via cross-streaking experiments. The volatile organic compounds produced by the isolates were also characterized by headspace gas chromatography-mass spectrometry (HS/GC–MS), to shed light on the vast resource of secondary metabolites represented by the medicinal plants-associated bacterial endophytes.
The bacterial strains used in this work were isolated from the endosphere of flowers, leaves, and stems of the officinal plant O. heracleoticum, as described in Semenzato et al. (2023) [32]. Strains are referred to as OH (O. heracleoticum), followed by the letters F, L, and S (for Flowers, Leaves, or Stems, respectively), and numbered (Table 1). The taxonomic affiliation of the bacteria was previously obtained via amplification and sequencing of the 16S rRNA coding gene; GenBank accession numbers are available in Semenzato et al. (2023) [32], except for strains OHF10 (OR880887), OHS9 (OR880888), and OHS20 (OR880889). The endophytes were stored in a 20% glycerol (453752, Carlo Erba, Milan, Italy) stock at –80 °C. They were grown on Tryptic Soy Agar (TSA, Oxoid LTD, Hampshire, UK) at 30 °C for 48 h.
OHF (Flowers) | OHL (Leaves) | OHS (Stems) | |||
Strain | Genus | Strain | Genus | Strain | Genus |
1 | Bacillus | 1 | Exiguobacterium | 2 | Bacillus |
2A | Bacillus | 2 | Bacillus | 3 | Pseudarthrobacter |
2B | Peribacillus | 4 | Pseudarthrobacter | 4 | Bacillus |
2C | Bacillus | 5 | Peribacillus | 5 | Bacillus |
2D | Lysinibacillus | 6 | Exiguobacterium | 6 | Acidovorax |
2E | Lysinibacillus | 7 | N.A. | 7 | Curtobacterium |
2G | Bacillus | 9 | Peribacillus | 8 | Bacillus |
3 | Bacillus | 10 | Arthrobacter | 9 | Pantoea |
4 | Peribacillus | 11 | Exiguobacterium | 10 | Pseudarthrobacter |
5 | Arthrobacter | 12 | Bacillus | 11 | N.A. |
6 | Bacillus | 14 | Arthrobacter | 12 | Curtobacterium |
7 | Priestia | 15 | Neobacillus | 14 | Arthrobacter |
9 | Priestia | 16 | Labedella | 16 | Pseudomonas |
10 | Arthrobacter | 17 | Roseomonas | 18 | Pseudomonas |
11 | Bacillus | 18 | Bacillus | 19 | Erwinia |
12 | Bacillus | 20 | Bacillus | 20 | Pantoea |
13 | Cytobacillus | 21 | Peribacillus | 23 | Pseudomonas |
14 | Peribacillus | 23 | Bacillus | 24 | Pseudomonas |
15 | Pseudarthrobacter | 24 | Arthrobacter | ||
16 | Bacillus | 25 | Bacillus | ||
17 | Arthrobacter | ||||
18 | Kocuria | ||||
19 | Variovax | ||||
20 | Bacillus | ||||
21 | Pseudarthrobacter | ||||
22 | Arthrobacter | ||||
23 | Roseomonas | ||||
24 | Pseudarthrobacter |
Abbreviations: N.A., not assigned.
Eleven strains of the Burkholderia cepacia complex (Bcc) belonging to four different species were selected on the basis of their origin, i.e., a clinical (Cystic Fibrosis patients, CF) or an environmental (ENV) one (Table 2). Each strain was grown on LB agar medium (NaCl 10 g/L, yeast extract 5 g/L, tryptone 10 g/L, agar 15 g/L, Oxoid LTD) at 37 °C for 48 h.
Genus | Strain | Antibiotic resistance | Origin |
B. cepacia | FCF3 | Cystic Fibrosis Patient | |
LMG 1222 | Environmental | ||
B. cenocepacia | FCF23 | Cystic Fibrosis Patient | |
LMG 16656 | Cystic Fibrosis Patient | ||
LMG 21462 | Cystic Fibrosis Patient | ||
LMG 24506 | Cystic Fibrosis Patient | ||
K56-2 | Cystic Fibrosis Patient | ||
LMG 19230 | Environmental | ||
B. multivorans | LMG 13010 | Cystic Fibrosis Patient | |
LMG 17588 | Environmental | ||
B. ambifaria | LMG 19182 | Environmental | |
CoNS | 5419 | FOX, DA, CIP, LEV, SXT, TIG | Food |
5318 | P, E, CN, FD | Human | |
5377 | P, TE, E, TEC | Hospital | |
5403 | P, E, TIG, TE | Human | |
5323 | P, TE, TIG, E, CN | Human | |
5321 | P, E, CN, AK, FD | Human | |
5284 | P, TE, E, CN, FD | Hospital | |
5285 | E, CN, CIP, LEV, FD | Hospital | |
5383 | P, FOX, TE, E, CN | Hospital | |
5396 | P, FOX, SXT, CN, FD | Human | |
P. aeruginosa | ATCC 27853 | FOX, K | |
5779 | TOB, CAZ, FEP, MEM | Environmental | |
4189 | AK, TOB, CIP, LEV, CAZ, FEP, MEM, IPM, PRL, TZP | Medical device | |
5234 | AK, CAZ, ATM, TZP, PRL, FEP, CN, IPM, MEM, LEV, CIP, TOB | Medical device | |
5245 | CAZ, ATM, PRL, FEP, CN, LEV, CIP, IPM, MEM, TOB | Medical device | |
7/4 | CAZ, FEP, MEM, LEV, ATM | Environmental | |
7/5 | CAZ, FEP, MEM, TOB, ATM | Medical device | |
7/3 | CAZ, ATM, FEP, MEM | Environmental | |
5009 | ATM, CAZ, CIP, CN, FEP, IPM, LEV, MEM, PRL, TOB, TZP | Medical device | |
5236 | AK, ATM, CAZ, CIP, FEP, IPM, LEV, MEM, TOB | Medical device | |
S. aureus | ATCC 25923 | P, NA | |
3428 | P, C, RD, FD, TE, TGC, LZD | Medical device | |
5788 | P, FOX | Human | |
3709 | DA, TE, E | Food | |
3710 | DA, TE, E | Food | |
4070 | P, DA, TE, E, CIP, LEV, DAP | Food | |
4168 | AMP, P, DA, SXT, TE | Food | |
4302 | P, FOX, SXT, DAP | Food | |
4691 | P, FOX, E, CN, CIP, LEV, DAP | Hospital | |
4708 | P, FOX, CN, VA, DAP | Hospital | |
K. pneumoniae | ATCC 700603 | CAZ, AMP, ATM, PRL, TE | |
4409 | AK, AMX, FEP, CTX, CAZ, CIP, ETP, IPM, MEM, TZP, SXT, TIG | Human | |
4412 | AMX, FEP, CTX, CAZ, CIP, ETP, IPM, MEM, TZP, SXT, TIG | Human | |
4417 | AK, AMX, FEP, CTX, CAZ, CIP, ETP, IPM, MEM, TZP, SXT, TIG | Human | |
4420 | AK, AMX, FEP, CTX, CAZ, CIP, IPM, MEM, TZP, SXT, CN | Human | |
4422 | AK, AMX, FEP, CTX, CAZ, CIP, ETP, IPM, MEM, TZP, SXT | Human |
Abbreviations: AK, Amikacin; AMX, Amoxicillin; AMP, Ampicillin; ATM, Aztreonam; CTX, Cefotaxime; CAZ, Ceftazidime; CIP, Ciprofloxacin; CN, Gentamicin; DA, Clindamycin; DAP, Daptomycin; ETP, Ertapenem; E, Erythromycin; FD, Fusidic acid; FEP, Cefepime; FOX, Cefoxitin; IPM, Imipenem; K, Kanamycin; LEV, Levofloxacin; MEM, Meropenem; NA, Nalidixic acid; P, Penicillin G; PRL, Piperacillin; SXT, Sulfamethoxazole/trimethoprim; TE, Tetracycline; TEC, Teicoplanin; TIG, Tigecycline; TOB, Tobramycin; TZP, Piperacillin/tazobactam; VA, Vancomycin.
The other 36 pathogenic strains used in this work were isolated from different sources (hospital devices, foods, patients, healthy subjects, and the environment) and were previously characterized for their resistance to multiple antibiotics, using the disk-diffusion method [37]. Staphylococcus aureus, Coagulase-Negative Staphylococci (CoNS), Pseudomonas aeruginosa, and Klebsiella pneumoniae strains were provided by the Applied Microbiology laboratory (Health Sciences Department, University of Florence, Italy), while the standard bacteria S. aureus ATCC 25923, P. aeruginosa ATCC 27853, and K. pneumoniae ATCC 700603 were obtained from Thermo Fisher Diagnostics S.p.A. All the strains were grown on TSA plates at 37 °C for 24 h.
Endophytes antibacterial activity against Bcc strains was evaluated through the cross-streaking method (Supplementary Fig. 1). Petri dishes with or without a septum separating the two compartments (to permit the growth of the tester and the target strains without any physical contact) were used. Tester strains (i.e., the O. heracleoticum endophytes) were streaked across one half of a TSA plate and grown at 30 °C for 48 h, to allow the synthesis of antibacterial compounds. Single colonies of each target strain were then suspended in 50 µL of a 0.9% NaCl w/v solution. Target strains belonging to the Bcc were then streaked perpendicularly to the tester strain and plates were incubated at 30 °C for a further 48 h. Additionally, Bcc strains were streaked on half of a Petri plate in the absence of the tester and were allowed to grow at 30 °C for 48 h (positive growth control). The antagonistic effect was evaluated as the reduction or absence of the target strains growth compared to control plates. The different inhibition levels were indicated as follows: complete (3), strong (2), weak (1), and absence (0) of inhibition.
Bacterial suspensions of the other MDR pathogenic strains were prepared as mentioned before and then streaked perpendicularly to the tester strain using an inoculation needle; plates were incubated at 37 °C for a further 24 h. Additionally, target strains were grown at 37 °C for 24 h in the absence of the tester (positive growth control). The antagonistic effect was evaluated as described above. The different inhibition levels were indicated as follows: complete (4), strong (3), moderated (2), weak (1), and absence of inhibition (0).
The Inhibition Score (IS) and Sensitivity Score (SS) were calculated for each
tester and target strain, respectively, as the sum of the values obtained in each
antagonism experiment; the total IS for each plant compartment and the total SS
for CF and ENV Bcc groups, and for each MDR target group were calculated as the
sum of all IS/SS, normalized by the number of tester/target strains per group
(NIS and NSS). The average value of inhibition towards each MDR target group was
calculated as the ratio between the sum of all IS and the number of the target
strains in each group (TIS
Phylogenetic trees were constructed in MEGA XI [39] by aligning, using the
Muscle algorithm, the 16S rRNA gene sequences of endophytic strains belonging to
the Bacillus and Arthrobacter-Pseudarthrobacter genera
with 35 type strains’ 16S rRNA gene sequences downloaded from the Ribosomal
Database Project (RDP), selecting those exhibiting a higher percentage of
identity with the endophytes sequences (
Biomass obtained from bacterial culture (30–100 mg) was collected in Head Space (HS) vials. Vials were sealed and immediately analyzed. HS vials were conditioned at 40 °C for 20 minutes before extraction. Bacterial volatile organic compounds (VOCs) produced by bacterial strains were extracted from the vial headspace and injected into the Gas chromatograph (GC). Headspace extraction was performed with a 2.5 mL Syringe-HS (0.64-57-R-H, PTFE, GERSTEL) conditioned and held at 40 °C from sample collection to injection. Splitless injection was used.
Gas chromatographic analysis was performed adapting a previously reported method
[41]. In detail, an Agilent 7000C GC (Agilent Technologies, Inc., Santa Clara,
CA, USA) system was used, equipped with a split/splitless injector, fitted with
an Agilent HP5-MS UI capillary column (30 m
We first checked the ability of the entire panel of bacterial endophytes to inhibit the growth of eleven bacterial strains belonging to the Bcc, bacterial strains known for their resistance to numerous conventional antibiotics. Bcc species are responsible for infections in Cystic Fibrosis (CF) patients, leading to severe health complications such as necrotizing pneumonia and sepsis, reducing patients’ survival rates [42]. Treatment of Bcc infections is challenging because of their intrinsic resistance to various antibiotics and their tolerance to antibiotic exposure, especially in biofilm formations [43]. Current therapeutic approaches lack evidence-based guidelines and often follow various antibiotic protocols. However, the complete eradication of Bcc infection remains difficult, prompting the exploration of alternative strategies, such as the identification of compounds able to enhance antibiotic activity by targeting resistance mechanisms, or the employment of alternative therapies such as quorum-sensing inhibitors, natural antimicrobial peptides, and specifically designed vaccines [44].
Data obtained are shown in Fig. 1, whose analysis revealed that almost all the endophytes (with few exceptions, see for instance Peribacillus sp. OHF10 and Arthrobacter sp. OHS14) were able to inhibit the growth of at least two Bcc strains, even though at a different extent: some of them were able to completely interfere with the growth of the entire panel of Bcc strains (Bacillus sp. OHF9, Cytobacillus sp. OHF13, and Pseudarthrobacter sp. OHS10), while others exhibited a moderate/low degree of inhibition. In general, O. heracleoticum-associated strains were much more able to reduce or inhibit the growth of Bcc strains isolated from CF patients (NSS = 115) than those with an environmental origin (NSS = 72). This was previously observed for medicinal plants-associated bacteria isolated from different plant compartments and rhizospheric soil of Origanum vulgare L., Lavandula angustifolia Mill., and Echinacea purpurea L. [45, 46, 47]. The most sensitive strain was FCF3 (SS = 153), while LMG1222 (SS = 56) was the most resistant. Although the two strains belong to the same species (B. cepacia), their behavior in response to the antagonistic activity of the endophytes was completely different, suggesting the relevant role of the strains origin in determining their resistance profiles [48]. This finding is quite interesting, and it is worth further investigation. In our opinion, CF Bcc isolates obtained from healthcare settings may have been exposed to specific antibiotics or antimicrobial agents, potentially influencing their resistance profiles. In contrast, ENV strains might face a different selective pressure, including exposure to various stressors and competition, which could influence their differential response to antibacterial compounds [49]. Over time, these strains may have developed mechanisms to survive in the presence of antimicrobial agents found in their natural habitat, leading to higher resistance levels compared to clinical isolates.
Cross streaking against Burkholderia cepacia complex (Bcc) strains using plates without septum. Different inhibition levels were indicated as follows: complete (3, red), strong (2, orange), weak (1, yellow), and absence of inhibition (0, white). Inhibition and sensitivity scores were calculated by adding all the values obtained for each tester or target strain, respectively. CF, cystic fibrosis patient; ENV, environmental origin.
The analysis of the NIS revealed that the flowers compartment hosted the community with the highest inhibitory potential against the Bcc strains, with a NIS of 21, compared to the leaves and stems compartments (NIS = 17). Concerning the single strains, the highest IS (from 33 to 26) were registered for strains belonging to the genera Arthrobacter (OHF5, OHF22, OHL24), Pseudarthrobacter (OHF15, OHF21, OHF24, OHS3, OHS10), Bacillus (OHL23, OHS8), Cytobacillus (OHF13), Peribacillus (OHF2B), Priestia (OHF7, OHF9), Pantoea (OHS9), and Curtobacterium (OHS12). It is worth noting that the majority of the most active strains are Gram-positive, belonging to the Phyla Firmicutes and Actinobacteria, except for Pantoea sp. OHS9. It is widely recognized that the phylum Actinobacteria is responsible for over half of the natural bioactive compounds documented in literature surveys, encompassing antibiotics, immunosuppressive agents, antitumor agents, and enzymes [50]. They can be isolated from various environmental sources, such as soil samples, plants, and the marine environment, and in many cases, their potential to attenuate and/or inhibit the growth of Gram-negative and Gram-positive human pathogenic strains was documented [51, 52]. Antibacterial activities were also reported for the Phylum Firmicutes, especially for isolates belonging to the genus Bacillus [53, 54]. A previous work on bacterial endophytes isolated from O. vulgare L. reported similar results; indeed, the most active endophytic strains were all Gram-positive (with few exceptions), many of which belonged to the genera Arthrobacter and Bacillus [45]. Consistently, the most active bacterial endophytes associated with E. purpurea rhizospheric soil were affiliated to the genus Arthrobacter [47]. From these first observations, we can hypothesize that the different antibacterial patterns observed for O. heracleoticum-associated bacterial strains seem not related to the ecological niche from which they were isolated but might be linked to their taxonomical affiliation at the genus level.
The same results are also reported in Fig. 2. In this case, the tester and target strains were sorted based on hierarchical clustering (dendrograms on the top and the left of the heatmap), highlighting well-defined groups of strains.
Heatmap representation of the different inhibitory activity of O. heracleoticum endophytic strains against the Bcc group (without septum). Rows represent the different endophytic testers, while columns are the target strains. Cells color is based on the observed reduction or inhibition of target growth. Both columns and rows are ordered based on hierarchical clustering (dendrograms).
Target strains (columns) were divided into three clusters: the first one, on the left, groups the most sensitive strains, all of clinical origin (B. cepacia FCF3, B. cenocepacia LMG21465, and LMG506); the middle one includes three CF strains and one ENV strain (B. ambifaria LMG19182), which were strongly or completely inhibited by around 60% of tester endophytic strains; finally, the last group (on the right of the heatmap) is mostly composed of ENV strains, except for B. multivorans LMG13010, and include the most resistant Bcc strains. Hence, the hierarchical clustering confirmed that environmental strains are generally more resistant than clinical isolates (p = 0.02, ANOVA test).
Concerning the endophytes’ clustering (rows), the first group is formed by the most active tester strains, already listed in the previous paragraph; the second group consists of strains that were able to strongly or completely inhibit Bcc strains included in the first two target clusters; the third one groups five endophytes with very low or no antagonistic effect on Bcc strains; lastly, the fourth group includes strains with low IS.
For each endophyte, the taxonomic affiliation at the genus level and the
compartment of origin were color-coded on the left of the heatmap. 56.3% of
endophytic strains belonging to the first group were isolated from the flowers
compartment (9 out of 16 strains), while only two strains (12.5%) belonged to
the leaves-associated endophytic community. The nine OHF strains belong to the
genera Arthrobacter, Pseudarthrobacter, Bacillus,
Cytobacillus, Peribacillus, and Priestia, the latter
four all belonging to the family Bacillaceae. On the other hand, 4 out of 5
endophytes (80%) of the third group were associated with the stems compartment
and belonged to the genera Pseudomonas, Bacillus, and
Arthrobacter. From these first observations, we can hypothesize that the
different antibacterial patterns observed for O.
heracleoticum-associated bacterial strains seem not related to the ecological
niche from which they were isolated but might be linked to their phylogenetic
relatedness. For example, Pseudarthrobacter sp. OHF24 and OHS10,
isolated from flowers and stems, respectively, have similar activity against Bcc
target strains and belong to the same species [32]. Indeed, the ANOVA test
(Supplementary Table 1) confirmed that the IS obtained for each
endophytic strain was significantly related to the taxonomic affiliation of the
strain at the genus level (p
To ascertain which of these two parameters, i.e., the “ecological niche” or “taxonomic affiliation”, is strongly associated with the ability of endophytes to inhibit Bcc strains, our attention focused on the two main phylogenetic subgroups: the first is composed by 13 strains belonging to the genera Arthrobacter and Pseudarthrobacter, while the second one includes 16 strains belonging to the genus Bacillus. For each of the two subgroups, a phylogenetic tree based on 16S rRNA gene sequences and a heatmap portraying cross-streaking results against Bcc strains were obtained (Figs. 3 and 4).
Phylogeny and antagonistic interactions within the genera Arthrobacter and Pseudarthrobacter. (A) Phylogenetic tree for the genera Arthrobacter and Pseudarthrobacter. (B) Heatmap representation of the different inhibitory activity of endophytic strains belonging to genera Arthrobacter and Pseudarthrobacter against the Bcc group (without septum). Rows represent the different testers, while columns are the target strains. Cells color is based on the observed reduction or inhibition of target growth. Both columns and rows are ordered based on hierarchical clustering (dendrograms).
Phylogeny and antagonistic interactions within the genus Bacillus. (A) Phylogenetic tree for the genus Bacillus. (B) Heatmap representation of the different inhibitory activity of endophytic strains belonging to the genus Bacillus against the Bcc group (without septum). Rows represent the different testers, while columns are the target strains. Cells color is based on the observed reduction or inhibition of target growth. Both columns and rows are ordered based on hierarchical clustering (dendrograms).
Both subgroups include strains with (very) dissimilar inhibition patterns isolated from different plant organs. Concerning the Arthrobacter subgroup heatmap (Fig. 3B), tester strains, sorted based on hierarchical clustering, were divided into 5 clusters of strains, formed by (i) OHS10, OHF24, and OHL24, which exhibited the strongest antibacterial activity towards both CF and ENV Bcc strains; (ii) OHS3, OHF22, OHF21, OHF5, and OHF15, which were slightly less efficient against ENV Bcc strains; (iii) OHF17, OHL4, and OHL10, with reduced anti-Bcc activity compared to the previous two groups; (iv) OHL14 strain, which was able to interfere with the growth of Burkholderia strains FCF3, LMG21462, LMG24506, and LMG16656 only; (v) OHS14, with no antibacterial activity. Concerning the Bacillus subgroup (Fig. 4B), the 4 clusters are composed of (i) OHS8 and OHL23, which strongly or completely inhibited the growth of all target strains; (ii) OHF16, OHF1, OHL12, OHF2C, OHL2, OHS5, OHF2A, and OHF3, which were active only towards clinical target strains; (iii) OHF11 and OHS18, whose antagonistic effect was limited to Burkholderia strains FCF3, LMG21462, and LMG24506; (iv) OHF20, OHS4, OHF6, and OHS2, which have no or low antagonistic effect.
As can be seen from the annotation on the left of the heatmaps, there is no
evident clustering of strains based on the compartment of origin (p
The observed antagonistic activity of endophytes might be attributed to the production of diffusible but also volatile molecules with antibacterial activity. Since volatile organic compounds (VOCs) are involved not only in inter/intra-species communication but also in antagonism between microbes [56], the production of VOCs was analyzed on a smaller subset of 12 endophytes, all clustered in the first group of the heatmap (Fig. 2), isolated from all three compartments, and belonging to the genera Arthrobacter (OHF5, OHF24, and OHL24), Pseudarthrobacter (OHF15, OHF21, OHS3, and OHS10), Bacillus (OHL23 and OHS8), Priestia (OHF7 and OHF9) and Curtobacterium (OHS12). The cross-streaking tests were performed on Petri dishes divided into two halves by a physical septum, not allowing the diffusion of antibacterial molecules from the tester to the target strains. The obtained results are reported in Fig. 5.
Cross streaking against Bcc strains using plates with septum. Different inhibition levels were indicated as follows: complete (3, red), strong (2, orange), weak (1, yellow), and absence of inhibition (0, white). Inhibition and sensitivity scores were calculated as the sum of all the values obtained for each tester or target strain, respectively.
All the selected strains, apart from Curtobacterium sp. OHS12, were able to strongly or completely antagonize the growth of CF and ENV Bcc strains. Due to the presence of the physical septum, the antibacterial activity of these endophytes could be attributed solely to the production of VOCs. Also in this case, the antibacterial effect was stronger towards CF targets but still substantial against ENV Bcc strains. Overall, the inhibition patterns and the IS obtained in this experiment were the same or slightly lower than the ones obtained using plates without septum. However, three strains exhibit interesting discrepancies with respect to the IS shown in the cross-streaking experiments performed with Petri dishes without a septum. The two Priestia strains (OHF7 and OHF9), exhibiting an IS of 29 and 33, respectively, in the previous experiments, showed a decreased IS, which is particularly marked for Bcc strains of environmental origin. The third strain, Curtobacterium sp. OHS12, did not affect the growth of Bcc strains through the production of VOCs.
The most active strains, scoring an IS of 30, were Arthrobacter sp. OHF24 and OHL24, isolated respectively from the flowers and the leaves compartment and not close phylogenetically (Fig. 3A). In a previous work, the antibacterial potential of VOCs produced by a panel of 8 endophytic strains isolated from O. vulgare was evaluated against 10 Bcc strains [41]. Also in this case, the most active strain, OVS8, belonged to the genus Arthrobacter, which was also able to interfere with the growth of human MDR Klebsiella pneumoniae strains through the production of VOCs [57]. Within the Actinomycetes phylum, research studies mainly focused on the genus Streptomyces, which is widely known for its remarkable ability to produce a wide array of antibiotics and other bioactive compounds and thus employed in various medical, agricultural, and industrial applications [58]. Indeed, there is still limited evidence regarding the antibacterial compounds synthesized by Arthrobacter species, with most studies focusing, to date, on strains isolated from Antarctic or marine environments [51, 59, 60]. The results here obtained suggest the potentiality of Arthrobacter strains isolated from medicinal plants as a promising source of antibacterial molecules of diffusible and volatile nature.
The endophytic strains showing the highest inhibitory activity against Bcc strains were also tested against 36 MDR human pathogens, belonging to S. aureus, P. aeruginosa, K. pneumoniae, and CoNS groups. The strains were selected because of their resistance to multiple antibiotics (Table 2). Moreover, the interest in these species is validated by their inclusion in the AR-ISS program in Italy, which actively participates in the European Antimicrobial Resistance Surveillance Network (EARS-Net), overseen by the European Centre for Disease Prevention and Control (ECDC). The primary goal of AR-ISS surveillance is to describe the antibiotic resistance within a specific group of pathogens isolated from invasive infections belonging to eight species (Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter species), to provide insights into the current status of antibiotic resistance and contribute in global efforts to address this growing concern in healthcare [61]. The same pathogenic species were previously employed in cross-streaking experiments aimed at demonstrating the antibacterial potential of O. vulgare bacterial endophytes; some of them, belonging to the genera Bacillus and Arthrobacter, were mostly active towards CoNS and S. aureus strains [45].
The antibacterial activity of the target strains was evaluated qualitatively, as
described in the previous paragraphs. Data shown in Fig. 6 revealed that the
selected endophytes reported the highest antimicrobial activity against the CoNS
group (except for strain 5419, isolated from a food sample), with a NSS = 27,
followed by K. pneumoniae (NSS = 26) and S. aureus (NSS = 16)
groups. On the contrary, P. aeruginosa strains were the most resistant
(NSS = 8). Interestingly, the most inhibited strains belonging to the CoNS group
are the ones of human origin, followed by strains isolated from hospital devices.
This is quite relevant considering the potential applications of the 12
endophytic strains as antibiotics producers able to face the spread of MDR human
pathogens. As verified by the ANOVA analysis, MDR target strains’ sensitivity was
significantly related to both taxonomic affiliation and isolation source
(p
Cross streaking against multidrug resistant (MDR) pathogenic strains using plates without septum. Different inhibition levels were indicated as follows: complete (4, deep red), strong (3, red), moderate (2, orange), weak (1, yellow), and absence of inhibition (0, white). Inhibition and sensitivity scores were calculated by adding all the values obtained for each tester or target strain, respectively.
Contrary to what was observed for the Bcc group, the flowers compartment hosted the community with the lower inhibitory potential, with a NIS of 45, compared to the leaves (NIS = 58) and stems compartments (NIS = 50). The highest IS was reported for the tester strain Bacillus sp. OHS8 (IS = 80), which was able to completely inhibit the growth of almost all the CoNS strains and induced a strong-moderate inhibition against K. pneumoniae and S. aureus groups. Apart from OHS8, the highest IS (from 56 to 66) were registered for strains belonging to the genera Pseudarthrobacter (OHF21, OHF24, OHS3), Arthrobacter (OHF5, OHL24), and Bacillus (OHL23). In particular, Bacillus sp. OHL23 was the only endophyte able to strongly inhibit the growth of all the S. aureus strains. On the other hand, the lowest antibacterial activity was reported for the two Priestia strains (OHF7 and OHF9), which had almost no antagonistic effect on P. aeruginosa and S. aureus groups.
Based on these results, 10 endophytic strains were tested for their ability to produce VOCs capable of antagonizing the growth of the 36 MDR human pathogenic strains, using Petri dishes with a septum physically separating the plate into two compartments, as described in the Materials and Methods section. Data obtained are shown in Fig. 7.
Cross streaking against MDR pathogenic strains using plates with septum. Different inhibition levels were indicated as follows: complete (4, deep red), strong (3, red), moderate (2, orange), weak (1, yellow), and absence of inhibition (0, white). Inhibition and sensitivity scores were calculated by adding all the values obtained for each tester or target strain, respectively.
In general, O. heracleoticum endophytes antibacterial activity was lower compared to the previous cross-streaking test results. Tester strains VOCs exhibited a moderate antagonistic effect towards the CoNS strains (NSS = 15), especially against S. epidermidis 5318 and 5403, both of human origin. The highest IS, ranging from 42 to 39, were registered for strains belonging to the genera Arthrobacter (OHF5, OHL24) and Pseudarthrobacter (OHF24).
Concerning the cross streaking performed against MDR pathogenic strains, the
total inhibition score (TIS) and the average value of TIS for each target group
(
Genus | Strain | CoNS | K. pneumoniae | P. aeruginosa | S. aureus | TOT | TOT | ||||
TIS | TIS | TIS | TIS | TIS | |||||||
Arthrobacter | OHF5 | 21 | 2.1 | 16 | 2.7 | 16 | 1.6 | 6 | 0.6 | 59 | 1.7 |
Cytobacillus | OHF13 | 12 | 1.2 | 15 | 2.5 | 7 | 0.7 | 3 | 0.3 | 37 | 1.2 |
Priestia | OHF7 | 9 | 0.9 | 5 | 0.8 | 2 | 0.2 | 1 | 0.1 | 17 | 0.5 |
Priestia | OHF9 | 14 | 1.4 | 2 | 0.3 | 3 | 0.3 | 0 | 0.0 | 19 | 0.5 |
Pseudarthrobacter | OHF15 | 17 | 1.7 | 16 | 2.7 | 12 | 1.2 | 9 | 0.9 | 54 | 1.6 |
Pseudarthrobacter | OHF21 | 24 | 2.4 | 16 | 2.7 | 14 | 1.4 | 12 | 1.2 | 66 | 1.9 |
Pseudarthrobacter | OHF24 | 19 | 1.9 | 16 | 2.7 | 15 | 1.5 | 10 | 1.0 | 60 | 1.8 |
Arthrobacter | OHL24 | 21 | 2.1 | 14 | 2.3 | 7 | 0.7 | 14 | 1.4 | 56 | 1.6 |
Bacillus | OHL23 | 28 | 2.8 | 1 | 0.2 | 30 | 3.0 | 0 | 0.0 | 59 | 1.5 |
Bacillus | OHS8 | 38 | 3.8 | 15 | 2.5 | 20 | 2.0 | 7 | 0.7 | 80 | 2.3 |
Pantoea | OHS9 | 11 | 1.1 | 6 | 1.0 | 10 | 1.0 | 0 | 0.0 | 27 | 0.8 |
Curtobacterium | OHS12 | 17 | 1.7 | 9 | 1.5 | 5 | 0.5 | 5 | 0.5 | 36 | 1.1 |
Pseudarthrobacter | OHS3 | 23 | 2.3 | 11 | 1.8 | 13 | 1.3 | 10 | 1.0 | 57 | 1.6 |
Pseudarthrobacter | OHS10 | 20 | 2.0 | 13 | 2.2 | 10 | 1.0 | 7 | 0.7 | 50 | 1.5 |
Genus | Strain | CoNS | K. pneumoniae | P. aeruginosa | S. aureus | TOT | TOT | ||||
TIS | TIS | TIS | TIS | TIS | |||||||
Arthrobacter | OHF5 | 16 | 1.6 | 5 | 0.8 | 9 | 0.9 | 9 | 0.9 | 39 | 1.1 |
Pseudarthrobacter | OHF15 | 10 | 1.0 | 3 | 0.5 | 6 | 0.6 | 8 | 0.8 | 27 | 0.7 |
Pseudarthrobacter | OHF21 | 16 | 1.6 | 1 | 0.2 | 6 | 0.6 | 10 | 1.0 | 33 | 0.9 |
Pseudarthrobacter | OHF24 | 14 | 1.4 | 4 | 0.7 | 12 | 1.2 | 12 | 1.2 | 42 | 1.1 |
Bacillus | OHL23 | 14 | 1.4 | 1 | 0.2 | 2 | 0.2 | 4 | 0.4 | 21 | 0.6 |
Arthrobacter | OHL24 | 15 | 1.5 | 0 | 0.0 | 5 | 0.5 | 20 | 2.0 | 40 | 1.0 |
Pseudarthrobacter | OHS3 | 18 | 1.8 | 2 | 0.3 | 3 | 0.3 | 6 | 0.6 | 29 | 0.8 |
Bacillus | OHS8 | 20 | 2.0 | 3 | 0.5 | 4 | 0.4 | 6 | 0.6 | 33 | 0.9 |
Pseudarthrobacter | OHS10 | 15 | 1.5 | 4 | 0.7 | 5 | 0.5 | 8 | 0.8 | 32 | 0.9 |
Curtobacterium | OHS12 | 12 | 1.2 | 1 | 0.2 | 4 | 0.4 | 6 | 0.6 | 23 | 0.6 |
The VOCs produced by the most interesting strains were analyzed using HS-GC/MS. The identified VOCs are listed in Table 5. Results are expressed as the area of each peak in the chromatograms normalized by the bacterial biomass. Headspace-GC (HS-GC/MS) was chosen since it reduces sample manipulation, does not require the use of solvents, and because of its easy preparative steps [41]. A total of 16 distinct metabolites were detected, mainly belonging to the following structurally distinct classes: alcohols (1-Butanol, 1-Butanol-3-methyl, 1-Hexanol, 2-ethyl-, 2-Propanol), ketones (2-Butanone, 2-Butanone, 3-methyl-, Acetone), hemiterpenes (isoprene), and sulphurated compounds (Bis(methylthio) methane, Carbon disulfide, Dimethyl sulfide, Dimethyl disulfide, Dimethyl trisulfide, Ethanethioic acid S-methyl ester, Metanthiol, Thiophene) (see Table 5).
1-Butanol | 1-Butanol-3-methyl | 1-Hexanol, 2-ethyl- | 2-Propanol | 2-Butanone | 2-Butanone, 3-methyl- | Acetone | Isoprene | Bis(methylthio) methane | Carbon disulfide | Dimethyl sulfide | Dimethyl disulfide | Dimethyl trisulfide | Ethanethioic acid, S-methyl ester | Metanthiol | Thiophene | |
Arthrobacter sp. OHF5 | 4.33 | 7.07 | 18.36 | 13.74 | 3.91 | 2.83 | 6.92 | 5.04 | 0.00 | 2.29 | 13.20 | 17.32 | 0.29 | 0.00 | 0.00 | 4.68 |
Priestia sp. OHF7 | 2.05 | 0.00 | 0.00 | 0.90 | 1.39 | 0.10 | 94.70 | 0.14 | 0.00 | 0.07 | 0.31 | 0.26 | 0.00 | 0.00 | 0.00 | 0.09 |
Priestia sp. OHF9 | 1.78 | 0.92 | 0.00 | 1.94 | 1.12 | 0.09 | 93.27 | 0.13 | 0.00 | 0.08 | 0.31 | 0.24 | 0.00 | 0.00 | 0.00 | 0.10 |
Pseudarthrobacter sp. OHF15 | 3.92 | 6.63 | 13.20 | 23.26 | 4.09 | 1.69 | 7.85 | 5.33 | 0.00 | 1.32 | 17.12 | 12.58 | 0.17 | 0.00 | 0.00 | 2.86 |
Pseudarthrobacter sp. OHF21 | 5.26 | 6.51 | 16.22 | 23.27 | 6.45 | 2.15 | 7.32 | 2.87 | 0.00 | 1.61 | 15.27 | 9.83 | 0.25 | 0.00 | 0.00 | 3.00 |
Pseudarthrobacter sp. OHF24 | 4.94 | 7.35 | 17.00 | 18.96 | 5.96 | 3.70 | 10.71 | 4.63 | 0.00 | 0.76 | 13.84 | 7.78 | 0.23 | 0.00 | 0.00 | 4.14 |
Bacillus sp. OHL23 | 6.05 | 7.51 | 20.66 | 12.28 | 4.37 | 1.92 | 9.17 | 5.88 | 0.00 | 2.35 | 15.24 | 9.67 | 0.20 | 0.00 | 0.00 | 4.69 |
Arthrobacter sp. OHL24 | 0.36 | 0.30 | 7.58 | 0.00 | 0.78 | 0.19 | 0.63 | 0.29 | 0.05 | 0.72 | 35.59 | 51.41 | 0.11 | 0.14 | 1.72 | 0.15 |
Pseudarthrobacter sp. OHS3 | 7.45 | 6.04 | 25.77 | 14.41 | 6.85 | 2.63 | 12.99 | 3.18 | 0.00 | 1.08 | 8.62 | 7.13 | 0.23 | 0.00 | 0.00 | 3.63 |
Bacillus sp. OHS8 | 3.57 | 1.84 | 8.11 | 20.56 | 1.85 | 1.27 | 31.88 | 2.57 | 0.01 | 0.81 | 18.67 | 5.87 | 0.15 | 0.00 | 0.00 | 2.84 |
Pseudarthrobacter sp. OHS10 | 8.07 | 14.51 | 15.44 | 25.99 | 4.91 | 1.69 | 7.84 | 2.41 | 0.00 | 1.13 | 10.61 | 4.63 | 0.10 | 0.00 | 0.00 | 2.68 |
Among all strains, Arthrobacter sp. OHL24 exhibited the highest VOCs content based on the normalized area of all peaks, followed by Priestia strains OHF9 and OHF7 (Fig. 8).
Total Volatile organic compounds (VOCs) produced by each strain. Histogram bars represent the total normalized area of the chromatogram peaks.
Based on the relative peak area, the compound dimethyl disulfide was the most abundant volatile for Arthrobacter sp. OHL24. Among sulfides, dimethyl sulfide was produced by almost all strains. It was demonstrated that dimethyl disulfide induced significant growth inhibition of different microbial pathogens, such as Rhizoctonia solani and Pythium ultimum, or many Gram-negative and Gram-positive bacteria [62].
For Pseudarthrobacter strains OHF15, OHF21, OHF24, and OHS10,
2-propanol was the most abundant compound, while it was acetone for
Priestia sp. OHF7 and sp. OHF9, and Bacillus sp. OHS8. Notably,
Priestia strains OHF7 and OHF9 produced acetone as the main compound
(
The general antibacterial activity of the endophytic strains can be correlated to the production of thiophene, while the higher activity of Arthrobacter sp. OHL24 against S. aureus strains could be ascribed to the production of sulphurated compounds.
Overall, the present study highlighted that endophytic bacteria associated with the medicinal plant O. heracleoticum exhibited, although to a very different extent, the ability to antagonize the growth of the human opportunistic pathogens belonging to the B. cepacia complex. The different inhibition patterns observed are not related to the plant compartment from which the endophytes were isolated, but to their taxonomic classification at the genus level; this assumption has been confirmed with a deeper analysis at the species level focused on two of the most represented and most active bacterial genera, Bacillus and the Arthrobacter/Pseudarthrobacter group. Bcc strains showed different sensitivity to the endophytes, which was attributed to their origin (clinical strains are more sensitive than environmental strains). Cross-streaking tests against the 36 MDR human pathogens revealed the highest antimicrobial activity towards the CoNS and K. pneumoniae strains. Consistently, the strains of human origin were the most inhibited, in both target groups. On the contrary, P. aeruginosa strains isolated from the environment or medical devices showed the lowest susceptibility. Cross-streaking tests using plates with a septum confirmed the strains’ ability to produce VOCs with antimicrobial properties, able to induce a strong antagonist effect towards the Bcc strains and a moderate one against the CoNS strains of human origin. Sulphurated compounds were responsible for the prominent antibacterial activity of Arthrobacter sp. OHL24 against S. aureus strains. Moreover, strains Priestia sp. OHF7 and OHF9 were good ketones producers and could be considered for further biotechnological applications. Further tests to investigate non-diffusible metabolites with antibacterial activity are in due course. In conclusion, this study points up the diverse antagonistic capabilities of O. heracleoticum-associated endophytes against both Bcc and MDR human pathogens, shedding light on taxonomic and biochemical factors contributing to their antimicrobial activities. These findings hold important implications for investigating new sources of antibacterial compounds and comprehending the intricate relationships that exist between medicinal plants and endophytic bacteria.
The 16S rRNA gene sequences of the endophytic strains used in this work are available in GenBank (NCBI) under the accession numbers from ON979588 to ON979656, OR880887, OR880888, and OR880889.
RF and GS designed the research study. GS, AE, ABer, FB, VC, SA, and APP performed the experiments. CC, ABec, SB, AMP, APP, GE, and RF supervised the experimental work. SB provided the initial plant material. GS, FB, CC, and APP wrote the original draft. GS cured the visualization of data. ABer, SA, ABec, VC, AE, AMP, GE, SB, and RF reviewed and revised the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.
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This research received no external funding.
Given their role as Guest Editors of the journal, Renato Fani, Giovanni Emiliani, and Giulia Semenzato had no involvement in the peer-review of this article and have no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Graham Pawelec. All other authors declare no conflict of interest.
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