Elma L Leite

Alberto F De Oliveira

Fillipe L R Do Carmo

Nadia Berkova

Debmalya Barh

Preetam Ghosh

Vasco Azevedo

R Do Carmo

Bacteriocins as an alternative in the treatment of infections by Staphylococcus aureus

Keywords: Staphylococcus aureus, antibiotic resistance, bacteriocins, biotechnology use of bacteriocins

Staphylococcus aureus (S. aureus) is a highly versatile Gram-positive bacterium that is carried asymptomatically by up to 30% of healthy people, while being a major cause of healthcare-associated infections, making it a worldwide problem in clinical medicine. The adaptive evolution of S. aureus strains is demonstrated by its remarkable capacity to promptly develop high resistance to multiple antibiotics, thus limiting treatment choice. Nowadays, there is a continuous demand for an alternative to the use of antibiotics for S. aureus infections and a strategy to control the spread or to kill phylogenetically related strains. In this scenario, bacteriocins fi t as with a promising and interesting alternative. These molecules are produced by a range of bacteria, defi ned as ribosomally synthesized peptides with bacteriostatic or bactericidal activity against a wide range of pathogens. This work reviews ascertained the main antibiotic-resistance mechanisms of S. aureus strains and the current, informative content concerning the applicability of the use of bacteriocins overlapping the use of conventional antibiotics in the context of S. aureus infections. Besides, we highlight the possible application of these biomolecules on an industrial scale in future work.

INTRODUCTION

The Gram-positive bacterium Staphylococcus aureus (S. aureus) is immobile, non-sporeforming, produces coagulase, and is often unencapsulated or has a limited capsule [START_REF] Acedo | The expanding structural variety among bacteriocins from Gram-positive bacteria[END_REF][START_REF] Vestergaard | Antibiotic Resistance and the MRSA Problem[END_REF]. S. aureus is both a human commensal and opportunistic pathogen. Mammal's skin is the primary environment to the colonization of members of the Staphylococcus genus in which can facilitates infection in immunologically suppressed individuals [START_REF] Otto | Basis of virulence in communityassociated methicillin-resistant Staphylococcus aureus[END_REF][START_REF] Weisser | Hypervariability of Biofilm Formation and Oxacillin Resistance in a Staphylococcus epidermidis[END_REF]. Specifi cally, S. aureus is the main trigger of several diseases such as osteomyelitis [START_REF] Hatzenbuehler | Diagnosis and management of osteomyelitis[END_REF], septic arthritis [START_REF] Deesomchok | Clinical study of culture-proven cases of non-gonococcal arthritis[END_REF][START_REF] Ryan Mj | Bacterial joint infections in England and Wales: analysis of bacterial isolates over a four year period[END_REF][START_REF] Shirtliff | Acute septic arthritis[END_REF], bacteremia [START_REF] Mylotte | Prospective study of 114 consecutive episodes of Staphylococcus aureus bacteremia[END_REF][START_REF] Shurland S | Comparison of mortality risk associated with bacteremia due to methicillin-resistant and methicillin-susceptible Staphylococcus aureus[END_REF]), endocarditis [START_REF] Watanakunakorn | Infective endocarditis at a large community teaching hospital, 1980-1990. A review of 210 episodes[END_REF][START_REF] Cabell Ch | Changing patient characteristics and the effect on mortality in endocarditis[END_REF][START_REF] Petti | Staphylococcus aureus bacteremia and endocarditis[END_REF], pneumonia [START_REF] Watanakunakorn | Bacteremic Staphylococcus aureus pneumonia[END_REF][START_REF] González | Staphylococcus aureus bacteremic pneumonia: differences between community and nosocomial acquisition[END_REF][START_REF] De | Staphylococcus aureus bacteremic pneumonia[END_REF], and mastitis [START_REF] Tenhagen B-A | Prevalence of pathogens in milk samples of dairy cows with clinical mastitis and in heifers at first parturition[END_REF][START_REF] Deb R | Trends in diagnosis and control of bovine mastitis: a review[END_REF][START_REF] Gomes F & Henriques M | Control of Bovine Mastitis: Old and Recent Therapeutic Approaches[END_REF]. It is noteworthy that this bacterium is also the principal causal agent of numerous implant infections [START_REF] Arciola Cr | Implant infections: adhesion, biofilm formation and immune evasion[END_REF]). Bacterial infections have been treated by the use of antibiotics since the beginning of the '40s. Still, their effi ciency tends to decrease due to the high rate of antibiotic resistance developed by several microorganisms, such as S. aureus (Ventola 2015a). The inappropriate use of antibiotics, particularly their overuse, has been considered one of the factors contributing to multidrug resistance in bacteria. This constitutes a severe global public health problem since the frequency of established and emerging infectious diseases has increased because of the ineffectiveness of antibiotics. This issue brings us a warning about the risk of the inefficacy of our current drug arsenal and pushes us to look for new strategies in the struggle against bacterial infection (Ventola 2015b).

The World Health Organization (WHO) has been treating the case as a danger of worldwide alert since the mechanisms of resistance are emerging and spreading around the world faster than is usual. To prevent and control antibiotic resistance, the WHO advises the healthcare industry to invest in the development of new antibiotics and novel strategies to treat bacterial infections. At the same time, it is estimated that close to 80% of all antibiotics sold just in the United States are intended for animal use [START_REF] Martin Mj | Antibiotics Overuse in Animal Agriculture: A Call to Action for Health Care Providers[END_REF]. In 2015, it was estimated that the global average annual consumption of antibiotics per kilogram of animals produced using cattle, chicken, and pigs would increase by 67% from 63.151 ± 1.560 tons to 105.596 ± 3.605 tons [START_REF] Van Boeckel | Global trends in antimicrobial use in food animals[END_REF]. These data show that there is the uncontrolled use of antibiotics favoring an exponential multidrug resistance acquired by bacteria and associated with the challenges in the development of new antibiotics that make this situation alarming. In contrast to pathogenic microorganisms and their pathogenic factors, the small molecules produced by certain bacteria called bacteriocins appear to be a different strategy and strong candidates to overlap/replace the role of conventional antibiotics. Bacteriocins are mainly defined as proteins and peptides which inhibit the growth or kill another related and unrelated microorganisms (Klaenhamme 1988[START_REF] Balciunas | Novel biotechnological applications of bacteriocins: A review[END_REF][START_REF] Chikindas Ml | Functions and emerging applications of bacteriocins[END_REF][START_REF] Lopetuso Lr | Bacteriocins and Bacteriophages: Therapeutic Weapons for Gastrointestinal Diseases?[END_REF].

This review gathers recent information about the possible use of bacteriocins replacing antibiotic treatment during S. aureus infection, considering the advantages offered by these natural compounds, as well as the differences and comparisons between them.

MECHANISMS OF ANTIBIOTIC RESISTANCE IN S. aureus

S. aureus is a highly adaptive and versatile Gram-positive bacterium that can cause a wide range of infectious diseases in humans and animals. The success as a pathogen relies on the combination of diverse virulence factors, invasiveness, and antimicrobial resistance [START_REF] Le Loir | Staphylococcus aureus and food poisoning[END_REF][START_REF] Al | Evaluation of Approaches to Monitor Staphylococcus aureus Virulence Factor Expression during Human Disease[END_REF]. Besides, S. aureus is capable of surviving in different environmental conditions [START_REF] Al | Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions[END_REF]). The first case of resistance to penicillin by S. aureus was reported in mid of the '20s. After this, several cases of antibiotic resistance to these bacteria have been reported [START_REF] Humphreys | Ciprofloxacin-resistant Staphylococcus aureus[END_REF][START_REF] Kaiser | Pharmaceutical Innovation[END_REF][START_REF] Vt | The Quinolones: Past, Present, and Future[END_REF]. Table I shows a broad class of antibiotics resistance and the targets of the mechanisms of actions of antibiotics utilized by S. aureus. β-lactam resistance in S. aureus strains S. aureus can acquire resistance to β-lactam antibiotics like penicillin and methicillin [START_REF] Fuda Ccs | β-Lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome[END_REF]) (Figure 1). Penicillin, which was discovered by Alexander Fleming in 1929, was the first antibiotic used to fight S. aureus-mediated infections. Penicillin inhibits peptidoglycan formation (PG) cross-links in the bacterial cell wall, which generate a three-dimensional structure around the cell, and ensures bacterial integrity. Thus, the penicillin binds to An Acad Bras Cienc (2020) 92(Suppl. 2) e20201216 3 | 17 DD-transpeptidase, an enzyme responsible for the formation of PG cross-links, and prevents its catalytic activity, as part of PG synthesis is inhibited by penicillin, while that the hydrolases and autolysins, bacterial enzymes involved in PG, remain actives. Thus, the activity of penicillin that causes an imbalance between PG synthesis and degradation weakens PG and leads to cell death. [START_REF]On the Antibacterial Action of Cultures of a Penicillium, with Special Reference to their Use in the Isolation of B. influenzae[END_REF]. Only two years after its introduction, S. aureus penicillin-resistant strains appeared. These strains contained an enzyme, β-lactamase, able to destroy penicillin [START_REF] Dietz | The Susceptibility of Penicillinaseproducing Bacteria to Penicillin: II. The Effect of Sodium Azide[END_REF]. The blaZ gene with blaR1 and blaI genes are the genetic determinants of S. aureus resistance to penicillin. The code for the β-lactamase enzyme, the presence of penicillin sensor, and a transcriptional repressor of blaZ expression respectively, β-lactamase acts on penicillin outside the cell by changing its molecular structure to an inactive form, penicilloic acid by the hydrolysis of its β-lactam ring. The expression of blaZ is controlled by external penicillin availability. Penicillin interacts with the transmembrane protein BlaR1. This interaction leads to the autocatalytic activation of BlaR1 to BlaR2 (or BlaR1 in the active form) that can promote the inactivation of the BlaI repressor and, therefore blaZ to synthesize enzyme. The three genes are located on a transposable element of a large S. aureus plasmid called b-lactamase-encoding transposon Tn552 that it shows to be persistent over time and be geography spread.

Furthermore, it was described as well as carry cadmium resistance genes, which can strengthen the role of resistance by attributing greater persistence among strains that carry this genetic content [START_REF] Shearer | Major Families of Multiresistant Plasmids from Geographically and Epidemiologically Diverse Staphylococci[END_REF]. Like the other β-lactams, methicillin impends the synthesis of bacterial cell walls. It inhibits cross-linkage between the linear peptidoglycan polymer chains s, which composes a significant component of the cell wall of gram-positive bacteria by binding to and competitively inhibiting penicillinbinding proteins (PBPs). These PBPs molecules also are called transpeptidases (D-alanylalanine). The mechanism that describes the methicillin-resistance S. aureus (MRSA) proceeds Fluoroquinolone-resistant S. aureus S. aureus has been reported as resistant to the quinolone class of antibiotics [START_REF] Tanaka | Mechanism of quinolone resistance in Staphylococcus aureus[END_REF][START_REF] Oizumi | Relationship between mutations in the DNA gyrase & topoisomerase IV genes and nadifloxacin resistance in clinically isolated quinolone-resistant Staphylococcus aureus[END_REF][START_REF] Lowy Fd | Antimicrobial resistance: the example of Staphylococcus aureus[END_REF]. This resistance is achieved due to specific mutations in two chromosomal genes: grlA coding for a subunit of DNA topoisomerase IV, the primary quinolone target reported [START_REF] Ferrero | Cloning and primary structure of Staphylococcus aureus DNA topoisomerase An Acad Bras Cienc[END_REF], and gyrA coding for DNA gyrase A subunit (Figure 3). The two proteins are intimately associated with the over-lapping and opening of the double DNA strand during DNA replication. Specific mutations in grlB and gyrB also cause resistance to quinolones (Andriole 2005). Both proteins have also been described as having the B subunit (grlB and gyrB) intrinsically associated with resistance in S. aureus. These mutations stand out in a region known as the quinoloneresistance determining region (QRDR), which trigger several codon alterations in synonymous and non-synonymous amino acid mutations [START_REF] Tanaka | Mechanism of quinolone resistance in Staphylococcus aureus[END_REF]. Mutations reduce the affinity of the enzyme-DNA complex for quinolones.

Linezolid resistance in Staphylococci

Considering the oxazolidinone class, the resistance of S. aureus to linezolid has also An Acad Bras Cienc (2020) 92(Suppl. 2) e20201216 6 | 17

been observed for the last decade [START_REF] Besier | Linezolid Resistance in Staphylococcus aureus: Gene Dosage Effect, Stability, Fitness Costs, and Cross-Resistances[END_REF]. The main feature of the mechanism of action describes the linezolid interaction with the 50S subunit of prokaryotic ribosomes, which prevents initiation factors such as formylmethionyl-tRNA from acting to form the complex with the 30S subunit. This disruption precludes the formation of the 70S complex, which sequentially disrupts protein synthesis (Figure 4 Regarding all the information explored here, the broad spectrum of antibiotics capable of inhibiting bacterial growth has been gradually decreasing over the last 70 years. All mechanisms of the bacterial resistance to the action of antibiotics are embedded in the complexity of interactions among genes and its mobile elements but mainly considering the bacterial hosts too. Moreover, the misuse of antibiotics by humans intensifies the selection pressure, making the resistance struggle even harder.

Bacteriocins against S. aureus infections

Bacteriocins are molecules usually produced by bacteria that can be used with biopreservative applications [START_REF] Bali | Trends in utilization of agro-industrial byproducts for production of bacteriocins and their biopreservative applications[END_REF]) but mainly in an antibiotic role [START_REF] Egan | Bacteriocins: antibiotics in the age of the microbiome[END_REF]). These biomolecules can be differently classified over the Gram-positive and Gram-negative bacteria (Table II). Most of them have low molecular mass (from less than 5kDa to 90 kDa), high isoelectric point, and contain hydrophilic and hydrophobic regions [START_REF] Lopetuso Lr | Bacteriocins and Bacteriophages: Therapeutic Weapons for Gastrointestinal Diseases?[END_REF].

It is suggested that the primary function of bacteriocins regarding their killing ability is directly associated with maintaining the population around to reduce the number of nutritional competitors in the environment. The targets for bacteriocins may have a broad spectrum of action similar to those of antibiotics (Table III), blocking several biologically important phases to the cell. Some recently characterized bacteriocins appear to have a common mechanism of action in which they dissipate proton-motive force (MPF), with modifications in membrane potential (∆ψ) and H + concentration gradient (∆pH) which consequently lead to the Table II. Classification of bacteriocins produced by Gram-positive [START_REF] Bierbaum | Lantibiotics: mode of action, biosynthesis and bioengineering[END_REF][START_REF] Varella Coelho M | Bacterial Labionin-Containing Peptides and Sactibiotics: Unusual Types of Antimicrobial Peptides with Potential Use in Clinical Settings (a Review)[END_REF]) and Gram-negative bacteria [START_REF] Rebuffat | Bacteriocins from Gram-Negative Bacteria: A Classification?[END_REF].

Classification Features Subclasses

Gram-positive

Class I Small, heat-stable peptides (<5 kDa), containing modified amino acids (lanthionine, 3-methyl-lanthionine, dehydrated amino acids, S-aminovinylcystein, among others) formation of pores in the cytoplasmic membrane [START_REF] Cleveland | Bacteriocins: safe, natural antimicrobials for food preservation[END_REF][START_REF] Perez Rh | Circular and Leaderless Bacteriocins: Biosynthesis, Mode of Action, Applications, and Prospects[END_REF].

Concerning the increased rate of S. aureus resistance, bacteriocins show great potential as candidates that can overlap the function of a large number of antibiotics (Table III) [START_REF] Cabell Ch | Changing patient characteristics and the effect on mortality in endocarditis[END_REF][START_REF] Ceotto-Vigoder H | Nisin and lysostaphin activity against preformed biofilm of Staphylococcus aureus involved in bovine mastitis[END_REF].

Several studies have described the bacteriostatic activity of bacteriocins that inhibits the growth of S. aureus, as observed in (Table IV). One work (Varella Coelho et al. 2017) described the inhibitory activity of seven bacteriocins in 165 strains of S. aureus in cases of bovine mastitis, showing a potent inhibition by epidermin (>85%) and a medium inhibition by aureocin A53 (>67%) [START_REF] Varella Coelho M | Bacterial Labionin-Containing Peptides and Sactibiotics: Unusual Types of Antimicrobial Peptides with Potential Use in Clinical Settings (a Review)[END_REF], however, the combination of aureocin 70 and A53 showed a more significant inhibitory potential when compared to previous results (>91%). Still exploring cases of bovine mastitis, another study [START_REF] Je | Activity of bacteriocins synthesized by Bacillus thuringiensis against Staphylococcus aureus isolates associated to bovine mastitis[END_REF]) highlighted five bacteriocins derivate from Bacillus thuringiensis that were tested against 50 strains of S. aureus recovered from the milk of lactating cows. The results of the study presented data on the resistance of these strains to penicillin, dicloxacillin, ampicillin, and erythromycin. However, all strains were susceptible to the five tested bacteriocins, showing them to be useful as an alternative approach to control bovine mastitis. Currently, one bacteriocin has shown strong relevance in the treatment of bovine mastitis [START_REF] Ceotto-Vigoder H | Nisin and lysostaphin activity against preformed biofilm of Staphylococcus aureus involved in bovine mastitis[END_REF]. The bacteriocin lysostaphin shows a minimal inhibitory concentration of 3.9 to 50 µg ml-1 compared to the usually used bacteriocin nisin, which was 15.6 to 500 µg ml-1. This study concludes that treatment using lysostaphin alone or associated with nisin was efficient in promoting bacterial cell lysis. Other studies investigating the role of biofilm formation in methicillin-resistant S. aureus (MRSA) strains, which has been observed to be able to alternate the resistance phenotype and thus attenuate the virulence [START_REF] Pozzi | Methicillin Resistance Alters the Biofilm Phenotype & Attenuates Virulence in Staphylococcus aureus Device-Associated Infections[END_REF], noted the effects of three bacteriocins. In this study, nisin A showed the highest bactericidal activity against planktonic and biofilm cells, while Lacticin Q showed lower activity. However, the Nukacin ISK-1 just showed bacteriostatic activity against planktonic cells. Despite this, the results show that the bacteriocins used to stand out as potent molecules effective in the treatment of MRSA infections [START_REF] Okuda | Effects of bacteriocins on methicillin-resistant Staphylococcus aureus biofilm[END_REF].

Recently, cases of MRSA have been achieving prominence in the search for new bacteriocins. [START_REF] Jiang | Purification, Characterization, and Mode of Action of Pentocin JL-1, a Novel Bacteriocin Isolated from Lactobacillus pentosus, against Drug-Resistant Staphylococcus aureus[END_REF] observed the role of pentocin JL-1 bacteriocin showing effectiveness in both gram-negative and positive bacteria [START_REF] Jiang | Purification, Characterization, and Mode of Action of Pentocin JL-1, a Novel Bacteriocin Isolated from Lactobacillus pentosus, against Drug-Resistant Staphylococcus aureus[END_REF]. Moreover, the authors also explored the ability of this bacteriocin to target the cell membrane of MRSA strains leading to cell death. At the same time, another study devoted to the investigation of the pattern of bacterial strains et al. 2019). Several analysis has highlighted the fact that, in addition to resistance to high temperature and various chemical compounds, it is a potent antimicrobial efficient against MRSA and a strong candidate for higher-yielding and enhancement of bacteriocin production.

Hanchi and collaborator, in a different study trying to comprise the synergy of antimicrobial, a group of bacteriocins and some antibiotics were evaluated considering the two subtypes to understand the efficacy of bacteriocins vs. antibiotics, these results show the effectiveness of bacteriocins nisin Z, pediocin pa-1, duracin 61 and reuterin as well, evidenced inhibition of MRSA [START_REF] Hanchi | Inhibition of MRSA and of Clostridium difficile by durancin 61A: synergy with bacteriocins and antibiotics[END_REF]. However, in the set of antibiotics used, only vancomycin displays effects against MRSA. Evaluation of synergistic activities of antimicrobial agents was showed that the combination of duracin with nisin or pediocin are a strong strategy in the control of growth bacterial, thus demonstrating the important role of duracin 61A as an active bacteriocin against clinical drug-resistant MRSA [START_REF] Hanchi | Inhibition of MRSA and of Clostridium difficile by durancin 61A: synergy with bacteriocins and antibiotics[END_REF]. Staden and collaborators show that nisin F-loaded self-setting brushite cement can control infection with S. aureus Xen 36 in mice model for 7 days [START_REF] Van Staden Ad | Nisin F-loaded brushite bone cement prevented the growth of Staphylococcus aureus in vivo: Inhibition of S. aureus by nisin F in bone cement[END_REF]. This study shows that the possibilities are bright for the use of bacteriocins to control infections.

BACTERIOCINS AS A BIOTECHNOLOGY TOOL IN THE TREATMENT BY S. aureus INFECTIONS

Interest in bacteriocins, as well as their production, has been growing over the years due to their use in food preservation, which exhibits antimicrobial activity as being an alternative to the use of chemical preservatives. Given the vast amount of methods capable of purifying these biomolecules, two approaches have been gaining more interest and becoming more effective in purifying bacteriocins: the Aqueous two-phase system (ATPS) and the Aqueous micellar two-phase system (AMTPS) [START_REF] Jamaluddin | Novel approaches to purifying bacteriocin: A review[END_REF]. These systems can be formed by mixing in a solution with various components. ATPS are generally formed when two polymers that are inconsistent, i.e., polyethyleneglycol (PEG) and dextran or sodium sulfate, are diluted in water [START_REF] Peters Tj | Partition of cell particles and macromolecules: Separation and purification of biomolecules, cell organelles, membranes and cells in aqueous polymer two phase systems and their use in biochemical analysis and biotechnology[END_REF][START_REF]Aqueous Two-Phase Systems[END_REF][START_REF] Grilo | Partitioning in Aqueous Two-Phase Systems: Fundamentals, Applications and Trends[END_REF]. There are other types including, ionic liquids and short-chain alcohols. The result of purification from ATPS displays a higher yield (~70%) [START_REF] Md Sidek | Aqueous two-phase flotation for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10[END_REF]) compared to the conventional method, for example, the single gel filtration chromatography (~1.0%). Interest has grown around this approach in the academic world, and recent studies have shown better results when the PEG / salt-based ATPS type is used, reaching yield values around 93% [START_REF] Sabo S Da S | Bacteriocin partitioning ELMA L. LEITE et al. INSIGHT INTO BACTERIOCINS APPLICATION IN S. aureus[END_REF]. Besides, the AMTPS results trying to improve the nisin extraction in the presence of electrolytes, show the advances in using the approach compared to conventional methods [START_REF] Jozala Af | Aqueous Two-Phase Micellar System for Nisin Extraction in the Presence of Electrolytes[END_REF]). Hence, the potential of ATPS and AMTPS as primary recovery methods for bacteriocins from a complex fermentation broth can be explored on an industrial scale, considering the easy handling and speed in obtaining these biomolecules, compared to the production stages already developed in the research laboratory.

Recent research on fermented vegetable extract is a good strategy and new application in the use of bacteriocins [START_REF] Feng | Recent research process of fermented plant extract: A review[END_REF]. Receiving the name of Fermented Plant Extract (FPE), this approach -most commonly performed as plain An Acad Bras Cienc (2020) 92(Suppl. 2) e20201216 12 | 17 liquid manure or plant extract can be used as a tool that assists in obtaining active substances providing a diversity of health benefits [START_REF] Altay F | A review on traditional Turkish fermented nonalcoholic beverages: Microbiota, fermentation process and quality characteristics[END_REF][START_REF] Aj | Fermented beverages with health-promoting potential: Past and future perspectives[END_REF]. Some plants of the Labiatae family, which contain a diversity of herbs shown to have antimicrobial activity [START_REF] Mahboubi A | Total Phenolic Content and Antibacterial Activity of Five Plants of Labiatae against Four Foodborne and Some Other Bacteria[END_REF]. FEP is also described as being fermented microorganisms, which include yeast and bacteria [START_REF] Blandino | Cereal-based fermented foods and beverages[END_REF][START_REF] Manzanilla Eg | Effects of butyrate, avilamycin, and a plant extract combination on the intestinal equilibrium of early-weaned pigs[END_REF]. This gives scope for the conclusion on the specific use of bacteria and plants, which may constitute a synergy in the antimicrobial treatment and a better understanding of the control by bacteriostatic phenotypes. The approach describing a composition comprising a bacteriocin and an extract from plant is described in a patent of [START_REF] Coyne B | Composition comprising a bacteriocin and an extract from a plant of the Labiatae family[END_REF]. This kind of strategy has been gaining strength and evidencing in numerous cases where the interruption of bacterial growth appears to be ineffective due to the use of these vegetable broths in conjunction with specific microbiotas [START_REF] Al | Analysis of Antibacterial, Antioxidant, and In Vitro Methane Mitigation Activities of Fermented Scutellaria baicalensis Georgi Extract*[END_REF]. Besides, extracts of tea and soybean showed dose-dependent growth inhibition of pathogens (Zhao & Shah 2015). This study used phenolic-enriched milk (PEM), fermented with lactic acid bacteria (LAB) and ultra-filtered, concluding that multiple agents, such as bacteriocins secreted by LAB, may exhibit synergistic antibacterial activity. This evidence on the role of FPE reveals the importance of their applicability, not only considering their use for health, thus presenting a new approach in treatments for S. aureus infections, but also the production on an industrial scale, and amongst other considerations, the environment.

CONCLUSIONS

Our work concludes that bacteriocins are becoming increasingly important in the fight against infections by several microorganisms, especially S. aureus. The mechanisms of resistance to antibiotics explored here show the need to obtain new strategies to combat this pathogen. Several bacteriocins having a latent action spectrum against S. aureus are shown to be as effective as or even more so than conventional antibiotics have been disclosed herein. Besides, although bacteriocins have great potential for functional overlap of some antibiotics, it is noteworthy that even these biomolecules are so susceptible to bacterial resistance as well as the common antibiotics. Thus, we believe that the information discussed here is remarkably important for animal and human health, as well as to provide a means of production on an industrial scale and new possibilities for future applications such as therapeutic methods.

  in a similar way to Penicillin. However, several works have discussed that the mecA gene that is responsible for methicillin resistance may be a mobile genetic element (MGE), which confers the ability to respond to environmental stresses. β-Lactams triggers the autolytic activation of the intracellular metalloproteinase domain (MPD), which is controlled by the integral-membrane zinc-dependent sensor (MecR1) a sensor protein and a transcriptional repressor (MecI) in the operator region to the expression of mecA[START_REF] Peacock | Mechanisms of Methicillin Resistance in Staphylococcus aureus[END_REF].Vancomycin resistance in S. aureusVancomycin is classified as one glycopeptide antibiotic susceptible to S. aureus-resistance[START_REF] Walters Ms | Vancomycin-Resistant Staphylococcus aureus -Delaware, 2015[END_REF]. Currently, are known four S. aureus strains are identified as vancomycinresistant: Vancomycin-sensitive S. aureus (VSSA), Vancomycin-resistant S. aureus (VRSA), Vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA)[START_REF] Mcguinness Wa | Vancomycin Resistance in Staphylococcus aureus[END_REF]. Vancomycin alters the peptidoglycan density in the cell wall, being capable of interacting with it forming non-covalent hydrogen bonds, in the exposed D-D-Ala-D-Ala peptides[START_REF] Hanaki | Activated cell-wall synthesis is associated with vancomycin resistance in methicillinresistant Staphylococcus aureus clinical strains Mu3 and Mu50[END_REF], which inhibits cell wall synthesis in the VSSA strains. Therefore, the mechanism of resistance can be associated with the presence of an enterococcal plasmid (vanA) or by the transposition of elements related to (Tn1546)(Zhu et al. 2010). Thus, the affinity of vancomycin to the polypeptide is heavily reduced due to the vanA operon provided by the conjugation of the plasmid in the VRSA strains, which can produce the different polypeptides D-Ala-D-Lac (Figure2).

Figure 1 .

 1 Figure 1. Scheme of Penicillin/Methicillin resistance. The active penicillins can promote the cleavage of the transmembrane protein (BlaR1 or MecR1) that leads the inactivation of the repressor protein on the operator region, allowing the expression of blaZ/mecA that synthesize the proteins capable of breaking the β-lactam ring in the various penicillins forming penicillanic acid derivatives, also inactive (Lowy 2003).

Figure 2 .

 2 Figure 2. Scheme of Vancomycin resistance. The main factor in the resistance role of Vancomycin is the vanA operon acquired by conjugal transfer. Initially, it is controlled by vanS and vanR, which are sensitive to the presence of vancomycin and active within the transcription of the operon. The genes vanA, vanH, and vanX are functionally associated with D-Ala-D-Lac synthesis and are responsible for the vancomycin resistance phenotype. Finally, vanY is described as being associated with the peptidase function, which cleavage the D-Ala-D-Ala that already were attached. The function of vanZ is still unclear and is not entirely understood.

  )[START_REF] Swaney Sm | The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria[END_REF]). However, some cases of bacteria resistant to linezolid bearing point mutations at the specific targets in the 50S, more precisely in the 23S ribosomal portion, have been reported[START_REF] Vg | Linezolid Resistance in Sequential Staphylococcus aureus Isolates Associated with a T2500A mutation in the 23S rRNA Gene and Loss of a Single Copy of rRNA[END_REF][START_REF] Afşar | Linezolid-resistant Enterococcus faecium: the first G2576T mutation in Turkey[END_REF]. Furthermore, a new mechanism of linezolid resistance, which was first explored by the presence in a plasmid that included the gene cfr, was detected[START_REF] Schwarz S | Identification of a Plasmid-Borne Chloramphenicol-Florfenicol Resistance Gene in Staphylococcus sciuri[END_REF]. Several years ago, a novel variant of the phenicol resistance transposon Tn558 was detected on the plasmid pSCFS6 suggesting the ability of horizontal transfer between staphylococci[START_REF] Kehrenberg | IS21-558 Insertion Sequences Are Involved in the Mobility of the Multiresistance Gene cfr[END_REF]). The transcription of the cfr gene produces a methyltransferase protein, which catalyzes the methylation of 23S rRNA at position A2503 offering resistance to some antibiotics such as chloramphenicol, florfenicol, and clindamycin[START_REF] Kehrenberg | A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: methylation of 23S ribosomal RNA at A2503: Drug resistance by methylation of A2503 in 23S rRNA[END_REF], and later to linezolid[START_REF] Toh S-M | Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid: Linezolid resistance through ribosome modification[END_REF]).

Figure 3 .

 3 Figure 3. Scheme of quinolones resistance. (a) The fluoroquinolones can inhibit the topoisomerase IV and DNA gyrase, preventing remodeling of the DNA molecule when it undergoes torsion due to the DNA replication, leading to cell death. (b) Mutations on Topoisomerase and DNA gyrase avoid fluoroquinolones activity, which confer antimicrobial activity allowing bacterial growth.

Figure 4 .

 4 Figure 4. Mode of action of Linezolid. Description mechanism of the common peptides biosynthesis processes in prokaryotes. The complex formation of the 30S and 50S ribosomes to 70S formation and the inhibiting of protein biosynthesis by Linezolid action.

Table I .

 I Description of the key events on the developing antibiotic resistance by S. aureus and the target of the mechanism of action utilized by this bacterium.

	Drug	Year drug introduced	Years to reported of resistance	Mechanism of action
	Penicillin	1941	1943
	Methicillin	1961	1962
				Cell envelope
	Vancomycin	1956	1997
	Ceftaroline	2010	2011
	Daptomycin	1980	1988 (resistance rates in vitro)	Disruption of bacterial plasma membrane function
	Ciprofloxacin	1985	1990	DNA replication
	Linezolid	2000	2001	Protein synthesis

An Acad Bras

Cienc (2020) 92(Suppl. 2) e20201216 4 | 17

Table III .

 III Biological functions affected by the action of bacteriocins and antibiotics.

	ELMA L. LEITE et al.
	Targeted biological functions	Bacteriocins	Antibiotics
	Cell envelope	Nisin A, nukasin ISK-1, NAI-107	β-lactams, glycopeptides
	DNA Replication and Transcription	Microcin B17, colicins, carocin S2	Quinolenes
		Geobacillin I, bac-GM17, plantaricins,
		dysgalacticin, lactococcin, pediocin-
	Membrane perturbers	like bacteriocins, mesentericin Y105,	Lipopetides
		lacticin Q, nisin A, Uberolysin, AS-48
		Bacteriocin
	Protein Synthesis	Colicins, cloacin DF13	Aminoglycosides, tetracyclines, chloramphenicol, macrolides
			Benzamide derivade,
	Septum Formation	Garvicin A, lactococcin 972	N-heterocycles, phenols/
			polyphenols, carboxylic acids
	Metabolism	-	Sulfonamides

INSIGHT INTO BACTERIOCINS APPLICATION IN S. aureus

An Acad Bras Cienc (2020) 92(Suppl. 2) e20201216 10 | 17

Table IV .

 IV Role of bacteriocins on preventing and control of the S. aureus growth.

	that are capable of exhibiting antibacterial
	activity against MRSA revealed one bacteriocin-
	like protein with a molecular mass of ~10 kDa
	produced by P. aeruginosa TA6 strain (Arumugam
	Bacteriocins	Class	Organism source	Reference
	Aureocins A70, A53 and 215FN (aureus) Pep5, Epidermin K7 and Epicidin 280 (epidermidis)	II and I	Staphylococcus aureus and Staphylococcus epidermidis	(Lívio Varella Coelho et al. 2017)
	Morricin 269, Kurstacin 287,
	Kenyacin 404, Entomocin 420	II	Bacillus thuringiensis	(Barboza-Corona et al. 2009)
	and Tolworthcin 524
	Lysostaphin	III	Staphylococcus simulans biovar	(Schindler & Schuhardt 1964, Lívio Varella Coelho et al.
			staphylolyticus	2017)
	Epidermicin NI01		Staphylococcus epidermidis	(Sandiford & Upton 2012)
	Pediocina PA-1	II	Lactococcus lactis	(Rodrıǵuez et al. 2005)
	Nisin A, lacticin Q, and Nukacin ISK-1	I	Lactococcus lactis QU warneri ISK-1 5 and Staphylococcus	(Okuda et al. 2013)
	Lacticin 3147	I	Lactococcus lactis subsp. lactis	(Twomey et al. 2000)
	Enterocin CCM 4231	II	Enterococcus faecium CCM 4231	(Lauková & Czikková 1999)
			Enterococcus faecium
	E 50-52 and OR-70	II	NRRL B-30746 and Lactobacillus salivarius	(Svetoch et al. 2008, Hanchi et al. 2017)
			NRRL B-30514
	Duracin 61A and Reuterin		Enterococcus durans 61A and Lactobacillus reuteri	(Hanchi et al. 2017)
	Pentocin JL-1	I	Lactobacillus pentosus	(Jiang et al. 2017)
	TA6	II	Pseudomonas aeruginosa TA6	(Arumugam et al. 2019)
		An Acad Bras Cienc (2020) 92(Suppl. 2) e20201216 11 | 17

Acknowledgments and funding

The authors would like to acknowledge the INRA -Institut National de la Recherche Agronomique for the support of this work and CAPES -Brazil. This work was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and, Finance Code 001 and by grants from INRA -Institut National de la Recherche Agronomique, France.

An Acad Bras Cienc (2020)

Author contributions

EL and AO developing and writing of draft preparation; FC, NB, DB, PG writing and suggestion of draft preparation, reviewing and editing; VA supervision contributions and provided the final draft of the manuscript.


G Matten
email: gael.matten@femto-st.fr

M Ouisse

M Collet

S Karkar
email: sami.karkar@ec-lyon.fr

H Lissek
email: herve.lissek@epfl.ch

R Boulandet
email: romain.boulandet@epfl.ch

M Versaevel
email: marc.versaevel@safran-nacelles.com

DESIGN AND EXPERIMENTAL VALIDATION OF AN ACTIVE ACOUSTIC LINER FOR AIRCRAFT ENGINE NOISE REDUCTION

The use of acoustic liners in aviation industry is a quite common solution for reducing the engines acoustic emissions. Although the current solutions based on single or multilayer liners are efficient and compact for the mid and high frequencies, noise mitigation in the low frequencies would require large volumes, making the integration in the nacelle difficult. Moreover, the passive liners are tuned to attenuate fixed frequencies and are optimized for specific flights regimes. An active electroacoustic skin based on a distribution of loudspeaker and microphones is presented here. The acoustic impedance is controlled by an embedded electronic system and can be changed in real time. Compared to a conventional passive liner, it is shown that the resonance frequency of the active skin can be adjusted to better match the flight phase and that the performance is better at low frequency. An experimental campaign in a wind tunnel has been performed and is presented here.

INTRODUCTION

The reduction of noise pollution due to civil aviation has become a major challenge for aircraft manufacturers. The use of passive liners inside aircraft nacelles is commonly used to attenuate the acoustic emissions of aircrafts but the efficiency is not broadband and the attenuation in low frequencies is poor. Moreover, the optimum acoustic impedance of the liner evolves according to the flight phase. Active noise control strategies have already shown their effectiveness [START_REF] Collet | Active acoustical impedance using distributed electrodynamical transducers[END_REF][START_REF] Lissek | Electroacoustic absorbers: bridging the gap between shunt loudspeakers and active sound absorption[END_REF]. The solution proposed here lies in the use of an active coating for acoustic impedance control. A prototype of this system is presented hereinafter.

ACOUSTIC IMPEDANCE CONTROL

The active skin presented here is made of an arrangement of unit cells used to synthesize a target acoustic impedance. A unit cell is the assembly of a speaker (actuator), four microphones (sensors) and an electronic control stage. The strategy consists in estimating the pressure at the center of the cell by averaging the signal of the four microphones and to act on the velocity field by the action of the membrane of the speaker.

The equation of control has been established in [START_REF] Rivet | Broadband Low-Frequency Electroacoustic Absorbers Through Hybrid Sensor-/Shunt-Based Impedance Control[END_REF] and is based on the Thiele/Small speaker model [START_REF] Richard | Closed-Box Loudspeaker Systems-Part 1: Analysis[END_REF]:

H loc (p) = S d Bl 1 - M ms p 2 + R ms p + 1 Cms µ 1 M ms p 2 + S d R at p + µ 2 Cms (1)
With :

• p : Laplace variable;

• S d : Effective piston area;

• Bl : Electromechanical conversion factor ;

• M ms : Mass of the mobile assembly;

• R ms : Mechanical resistance;

• C ms : Compliance of membrane suspensions;

• R at : Target acoustic resistance of the mobile assembly;

• µ 1 , µ 2 ∈ [0; 1] : Control parameters.
This control law is used to alter the dynamics of the electrodynamic actuator and would idealy synthesize a purely resistive acoustic impedance when µ 1 = µ 2 = 0. For stability considerations, these parameters can never be null, which would be equivalent to cancel the mass and the compliance of the loudspeaker. However µ 1 controls the apparent mass of the speaker and µ 2 its compliance.

EXPERIMENTAL DEVELOPMENTS

Prototype of active skin

The prototype is made of thirty 50x50mm cells. Each cell is the assembly of a speaker and each corner is equipped with a microphone (see figure 1). The current through the speaker is MEDYNA 2017 25-27 Apr 2017, Sevilla (Spain) controlled by a Howland current source [START_REF] Alexander S Ross | Current source design for electrical impedance tomography[END_REF]. A micro-controller computes the equation driving the dynamic of the electric current in the speaker reel depending on the average measured pressure. The output current is updated at a rate of 50kHz. The computations are performed locally but all the cells are connected to an interface card through a serial bus. Thus the parameters of all the cells can be changed and local features (RMS pressure and RMS current) are accessible from a computer.

Power consumption

With the control off, the active skin has a power of 12.5W, including 0.2W per micro-controller. The electronics is able to feed each speaker with a current of 0.25A i.e. a power of 1.25W. The maximum electric power is then 50W. In practice, with a controlled noise of 110dB RMS, only 5mA are required per speaker so the overall power is 20W for 30 cells.

Wind tunnel

The active skin prototype has been wall mounted in the wind tunnel FDF of the Netherlands Aerospace Centre (NLR). The test bench is shown in figure 2. The difference of acoustic intensity between the upstream and the downstream reverberant rooms gives the IL (insertion loss). The IL evaluates the acoustic energy absorbed by the active skin. The acoustic source is placed in the upstream room (downstream configuration) or in the downstream room (upstream configuration). Tests have been run with air flow up to Mach 0.15. The flow noise has saturated the microphones for flows faster that Mach 0.15. The figure 3 shows the results with flow in downstream and upstream configuration and the results without flow for a target resistance of

CONCLUSION AND PERSPECTIVES

prototype presented here is an active skin able to synthesize specified acoustic impedances. It aims at replacing passive liners in aircraft nacelles and has been tested in a wind tunnel with airflow up to Mach 0.15. Insertion Loss up to 16 dB have been measured. The acoustic impedance of the skin is re-programmable in real time so adaptive strategies can be implemented to best match the phases of flight.

Figure 1 :

 1 Figure 1: Picture of the active skin prototype comprising 30 loudspeakers and 120 microphones.

ρc 2 .

 2 Mitigations up to 16 dB have been measured. The resonance frequency of the speaker is denoted f 0 . The flow does not affect significantly the efficiency.

Figure 2 .Figure 3 .

 23 Figure 2. Experimental validation in a wind tunnel (NLR,the Netherlands).

ACKNOWLEDGEMENTS

This work has been supported by the ENOVAL European project and has been done in cooperation with Labex ACTION (ANR-11-LABX-0001-01).