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    Microbial Pathogenesis 124 (2018) 152-162 Contents lists available at ScienceDirect Ee Microbial Pathogenesis journal homepage: www.olsovier.com/locate/micpath Antimicrobial potential of Alpinia purpurata lectin (ApuL): Growth inhibitory action, synergistic effects in combination with antibiotics, and antibiofilm 6% activity Gustavo Ramos Salles Ferreira’, Jéssica de Santana Brito’, Thamara Figueiredo Procépio’, Nataly Diniz de Lima Santos’, Barbara José Rocha Cardoso de Lima’, Luana Cassandra Breitenbach Barroso Coelho", Daniela Maria do Amaral Ferraz Navarro‘, Patricia Maria Guedes Paiva’, Tatiana Soares’, Maiara Celine de Moura’, ‘Thiago Henrique Napoledo™” ‘parame de agin, Congo de coin, Une Fell de Pera, Refe, Romanus, rash ™ cae de Teena aris do Nae Ref Bra MICeq were inactlated into Petr plates containing MHA or SDA, which were then incubated for 24 hat 37°C or 28°C, respectively. The MBC and MFC corresponded to the lowest concentration able to reduce the number of CFU by 99.9% regarding the inital inoculum. Each assay was conducted in triplicate ‘and three independent experiments were performed. 2.8. Flow eytomery The viability of microbial cells treated with ApuL. was evaluated using the Cell Viability Kit of BD Biosciences (San Jose, CA, USA) The isolates were incubated as described in section 24 with Apul. at the MICyo values (lectin sensitive isolates) or at 400 g/ml. (in the ease of isolates for which MICzy was not detected). The negative control was prepared by adding distilled water instead of Apul. For the postive control, cells were treated with 70% (1) isopropyl alcohol for 1 before the analysis. The samples were centrifuged (10,0003, 10min, 25°C) andthe cll pellets were washed thrice with 0.1 M PBS (pH 7.0). Next, 42M thiazole orange (TO, Sui) and 4.3 mM propidium iodide (I, Sul) were added to the samples, which were vortexed and in- ‘cubated for min at 25°C Next, $0 ofa Muorescent bead suspension (BD Liquid Counting Beads) was added, and the mixture was vortexed for 305. Data were acquired In a BD Accuri C6 cytometer (BD Biosciences) with an SSC threshold of 200 and stopped after gating 20,000 events for each sample. Analysis wae performed using the BD Accuri C6 Software. The results were presented as FI ws. FL3 dot plots land mean FL fluorescence (PI staining). 26, Microbial growth curves “The 24 growth curves ofthe bacteria and yeasts after exposure to diferent Apu concentrations (0.25 % Map, 0.5 MiCzp nd MICs) orn the absence of lectin (negative control), were established in 96- well mirotter plate according to the method stated by Gaidamashi and Van Staden [32]. To each well, 8p of inoculum (10 CFU/ml) ‘was added during the exponential growth phase plus 80 of ApuL at the cited concentrations and 40 uL of MHB or SDB. The plates were incubated at 37°C or 28°C and the OD, was measured every hour. 2.7, Braluation of protein leakage “The leakage of proteins from microbial clls was evaluated 2c cording to Ogundare [33]. The concentration of Apu was adjusted to the MIGso value for the respective isolate in a total volume of 1 mL. coeresponding, 9 $00 pl. of the medium (MHB oF SDB) containing the lectin and 100. ofthe inoculum (108 CFU/ml). For negative controls, the lectin was replaced by distilled water. The assays were maintained ina shaking incubator at 37°C of 28°C for 24h, After this period, the samples were centrifuged (800 xg: 10min, 25°C) and the supernatants were evaluated for protein concentration (28). The amount of leaked protein was caleulated by subtracting the protein content at ime zero from the content at the end of assay. The assays were performed in quadruplicate 28, Microscopy analysis “Three-dimensional images ofthe structute of microbial cells treated for not with Apul were obtained by scanaing electron microscopy (GEM). The microbial cells (1.2m; 10°CFU/ml) were incubated with MHB or SDR (0.6m) and ApuL (04mL) at the respective MICzg in Mili:Q water. The negative control was performed by adding Mill-O water instead of lectin. After incubation (24hhat 37°C or 28°C), the samples were centrifuged (300 x g; 10min, 25°C) andthe precipitated cells were washed three times in 0.1 M cacodylate buffer and fixed in 2.5% glutaraldehyde/4% paraformaldehyde/5 mM CaCl, in 0.1 M cx ‘odylate buffer pl 7.2 for 30 min at 28°C. After washing with the same buffer, the cells were allowed to adhere to stubs (6 127 mm, 9mm length Ted Pella In, Redding, CA, USA) and post-fixed for 1h with 196 ‘osmium tetroxide/0.8% potassium ferricyanide/'SmM CaCls in 0.1 M ‘cacodylate buffer, pt 7.2. The ells were dehydrated in graded acetone, critical point-dried with CO, coated with a 20 nm-thick gol layer, and observed with a Quanta 200F (FE! Company, Hisboro, OR, USA) Scanning electron microscope. Cells without treatment were Used a5 controls 2.9, Synergism assay The synergistic effects between ApuL and antibiotics were evaluated ‘Table2 Mba Patogoes 1262018) 152-162 Evalation of antimicrobial activity of ApuL and reference drugs (spared or in combination) against Stapocccus aes, Paeudemonas aero, Candida abicans and Candida porpsioss Sephyoawcs aues ae a i combioton a ict of combination ea ‘min oat ‘rain ‘enna 670 S400 25 Py a7 10s on Synz ‘reas er bas ons oon a5 Since, dona ogi Cee Ca i combiotion nc ‘ito bition hoa a heat hatin ‘PEA 22 24 ‘ 08 2 as Sierz ne pecs MCs Ca i ambition snc ‘tof bination hoa acne heat Pca nt eae 200 om ‘02 02s 1 ‘ae (le porepens ‘eo ae aot 16 ou — Mc: minimal inhibitory concentration, expres in y/mls SFC: Sum ofthe fractional inhibitory concentration index ND: nt determined. NC: nt eakeated using the method described by Pillai and Moellering [24]. Combina tions of ApUL. and oxacillin or ceftazidime were tested against anti bioticresistnt and standard S, aureus and P. aeruginosa isolates, spectively, and with Nuconazole against Candid species. The asay was performed in order to obtain a range of concentrations of Apu. and ‘commercial drugs that would allow simultaneous detection of syner- sgism, addition, indifference or antagonism. Each experiment corre- sponded to two rows of a 96-well mieroplate, where the antibiotic was ‘added (80 Lat 8 MICs.) tothe fourth well ofthe Fest row and serial twofold dilution in sterile MII-Q water was performed until the pe- ultimate well of the second row. Nest, APU. was added (80 Lat 4% Mi) 10 the penultimate well of the second row and two-fold ‘eral dilution was carried out inthe opposite direction until the fourth wel of the first row. The third ll ofthe frst rove contained only the ‘drug (4 MICjg) and the last well ofthe second row contained only ‘Apul. (4% MICiq), while the intermediate wells contained mixtures of the two agents at different ratios. MHB or SDB (40) was added t all wells ofthe plate and tothe fist well which contained 200 of culture ‘medium and corresponded to a sterility etrol. The second well cor- responded to the 100% growth control. Each plate was inoculated with microbial suspension (60 ul at 10° CFU/ml.) and incubated at 37°C or 28°C for 24h, ‘The evaluation of the interaction Between the diferent treatments was performed by determining the sum of fractional inhibitory con centration index (2F10), a8 follows: 2F1C = (Miao of Apul. in combi: nation/MICsp of ApuL alone) + (OICaa of antibiotic in combination MiCuo of antibiotic alone) The combinations were clasified as sy nergisic (EFIC=0.5), additive (ERIC > 0.5-1.0), indiferent GHIC > 1.0-= 40), or antagonistic (FIG > 4.0) [55] 2.10, Antbioflm assay Biofilm formation was assessed in sterile polystyrene 96-well mi ‘roplates by the crystal violet method [36] and the ability of Apul in preventing or reducing the biofilm formation was determined. In the ‘assay, 80 iL, of microbial suspension (10* CFU/ml. in 0.15M NaCl), ‘BOUL of Apul. (at different concentrations based on MICs fr each ieroorganism) in Mil-Q water, and 40 iL of MHB or SDB were added to each wel, Milli-Q water was used in place of Apul in 100% biofilm formation controls (non-treated cells). Bacterial growth was assessed by following the inerease in ODpoo after 24h of incubation at 37°C or 28°C, Nest, nonadherent cells were removed from the wells by washing three times with 0.15 MI NaCl. The biofilms were feed with absolute methanol solution for 20min, heatfxed at 50°C for 60min, and then stained with 0.4% (w/v) crystal volt for 25 mint 25 °C. The ‘unbound eryeal vole was removed with distilled water, the dye that bound tothe biofilm was solubilized with absolute ethanol (15 min), and absorbance was measured at S70nm. All experiments were per. formed in triplicate. Ampicilin, oxailin, and Nuconazole (at MICsp value) were used as reference drugs. 2.11. Statistical analysis ‘The data were expressed as the mean or the percent mean standard deviation (SD) and statistical diferences were determined ‘sing Tukey's range test; p value < 0.05 was considered statistically significant 2. Results and discussion In view of the increasing need for new antimicrobial agents, plant lectins have been evaluated for antibacterial and antifungal activities; in addition, the mechanisms of action have been studied. The present work reports the investigation of antimicrobial action of the lectin Apu. against non-resistant and drug resistant pathogens as well as possible synergistic effets with antibioti supplementation, /ApL was isolated by gel-filtration chromatography on Sephadex G- 75 columns and showed specifi HA of 195 witha purification factor of 124. The HA ability of Apul. was inhibited by the glycoproteins fetuin and ovalbumin. All of these results corroborate with data previously reported by Brito etal. [26 1, Antibacterial activity Apu showed bacteriostatic activity against the non-resistant (UFPEDA.02) and one (UFPEDA.672) of the three oxacilin-resistant Isolates of aureus (ble 2). A lower MICag value was obtained forthe ‘non-resistant isolate, which was also the only one for which bactericidal activity was detected (MBC of 200 g/mL). The viabllities ofS. aureus cells treated with Apul and untreated S. aureus cells was evaluated ‘sing TO to stain all ells and PI as a marker of unviable eels. The results can be seeming 1. Increased staining with Pl was detected for Mota Paton 1242018) 152-162 UrpeDA.02 aTec-8538) urpepa.s7o : a - “ae Boe “Et ois" : ope 5 i, an : 7 i ie ie ae aso Mae et = _ ‘ : ' = i i= i : ime 7 bee J so a Saal . . Je | i i a _. = UrPEDASTe 7 ea “Ee aie : : i, OtLr Mow ote rb 7 ret im pe A 3 tw. T tavo de : Hise. ix i = 4000 | a = e000 < = 20 Som. = = ° : Res re ateea nee Ase Fig. 1, Flow cytometry analysis ofthe cel viablty of enbloe resistant and non-existant Stophyococur ares oats treated with Apa the MIC (UFPEDA (02/ATCC-6585 and UFPEDA.672) of at 400ug/mL (UEPEDA.670 and UFPEDA-671) and their untreated countespas. Calls incubated in the absence of letn ‘comesponded tothe negative contel (NO. Isopropyl alobol (70% v/s) was used asthe pstive control (PC). The FL ¥s FL3 dt pots an be seen (ounting beads ‘re not shown), The bar chars display the mean orescence in the F1. chane! which corresponds to the saning by propidium fodide. Date are expressed a the mean * standard deviation (SD). Different ltrs indicate significant diferences(p < 005) between the treatments: UFPEDA2 isa non-ressant isolate. The resistance profiles of the other isolates ean be sen in Table UFPEDA.02 and UFPEDA.672 treated with ApuL at the MIC) value, with the mean ¥L3 Quorescence being higher (p < 0.05) than that detected for the negative control. The staining of unviable cells was 478. and 5.79old higher for UFPEDA.O2 and UFPEDA.672, respec- tively, than thet forthe negative control These results indicate that the growth inhibition was asociated with the impairment of cell viability ‘The Pl staining of lectin-treated (400 pg/ml) cell of the UFPEDA-670 and UFPEDA.671 isolates was only slightly higher (p < 0.05) than that for the negative control and was much lower (p < 0.05) than that for the positive contro; this agrees with the absence ofa growth inhibitory Mba Patogoes 1262018) 152-162 ; . a . ee 3 ad # i oESREEEE untested counterpart ells incuba in the akence of etn corresponded othe negative control (NC), Ipopylaleahol (70%, v4) was used a the posive ‘contol (PC). The FL vs FL. doe plots ean be seen (counting beads are not shown). The bar chars display the mean Mborescence In the FLS chanel, which ‘omesponds tothe staining by propidium iodide. Data are expeesed as the mean + standard deviation (SD). Different letters indicat significant diferences, {p = 0005) between the tesiments UFPEDA 416/ATCL-27853 isa non-esitant loate. The resistance profiles ofthe othe slates canbe seen in Tobie 1. effet of Apul that would be sufficient enough to determine its MICsy ‘Apul- did not inhibit the growth of the P. aeruginosa isolates in more than 50%, preventing the determination of MIC values, For the non- resistant (UFPEDA-416) and one multidrug resistant (UEPEDA-262) isolate the cell viability was not affected by the presence of Ap at @ ‘concentration of 400 g/mL (Pig. 2). In this figure, it ean also be also {sen that the mean FL3 fluorescence in lectin-reated UFPEDA-261 cells ‘was higher than that in the negative control and similar to that in the Positive control; however, the degree of cell viability impairment was ‘ot enough to reduce the culture growth by atleast 50%. The antimicrobial effects of lectins may involve their interaction ‘with microbial cell wall components such as techole and telcuronic ‘acids, peptidoglycans and lipopolysaccharides present in Gram-positive and Gram-negative bacteria [37]. The binding of lectins to these com Ponents and the access of them to membrane surfaces may tigger several effects such as growth inhibition, damage to cellular integrity, alteration of membrane permeability and nutrient uptake, induetion of oxidative stress, and damage to respiratory processes, for example 25,33) ‘Apul. showed differential action an the Gram-positive and Gram: negative species evaluated, as well as exerted distinct effects on non- resistant and resistant isolates of the same species. This may be ex: plained because, as mentioned above, the antibacterial activity of lec- tins i linked to their interaction with carbohydrates at microbial sur faces, which may be different among distinct isolates of the same bacterium [39] Indeed, the amplitude ofthe spectra of action differs among lectins. A lestin isolated from Cladonia vertiillars lichen, pre ‘sented antibacterial activity against both Gram positive and Gram-ne ative bacteria (Bacillus subi, Enterococcus facais, Bscherchia col, ‘Klebsiella preumoriae, and S, aureus) [40] and lectin from Eugenia ma lacensis showed high bacteriostatic and bactericidal effects against several species, being more effective against aureus [41]. On the ‘other hand, the lectin from Calliandra surinamenss leaf pinnalae (Casul) shoved bacteriostatic activity against Gram-positive Stapyio- 0.05) between treatment. In order to clarify if Apul. would be acting or not by damaging bacterial structure, three-dimensional images of cells treated with the MiCag were obtained by SEM (ig. 4). The images obtained for UF- PEDAO2 $, aureur cells showed almost no differences between the controls (ig. 4A) and the cell after treatment (Fig. 4B). However, we can observe that Apul. may have induced bacterial eolleycle arrest, since some cells appear not to have completed cell division. This sug sest that Apul. promote minor damages to the membrane of UFPEDA- (cells, suficient ro result in a higher PI staining but not to increase significantly protein leakage and to be visualized by SEM. Against the resistant isolate UFPEDA-672, the lectin also did not eause serous da ‘mage to the bacterial structure, but the treated eels presented prom: nences on their surfaces without alteration of cell morphology. These results reinforced the idea that Apu promote growth inhibition and {impair cell viability through different mechanisms on S. aureus isolates: there was no protein leakage and structural damage on non-resistant cells, but cell division seemed tobe impaired, whereas forthe UFPEDA. 672 isolate, changes involving cll surfaces and membranes occurred, the cell viability was more affected (higher increase of PI staining in comparison with control), and there were structural alterations and protein leakage 3.2, Antifungal activity Apul showed fungistatic activity against C alicans and C. para pslsis (Fable 2) but MC value was not detected (> 400 g/mL). The flow cytometry analysis revealed that at the MICs, APUL- significantly (p © 0005) increased the number of unviable C. albicans and C. gue Let Mba Patogoes 1262018) 152-162 Fig. 3. Growth curves (24 ieabaton) and protein leakage insets) from el of Saplylooccus aureus non-resistant plate UFPEDA 02 (A) and oxen resistant inolate UFPEDA.672(B) as well of the yeas Candid alban (C) and Candida porapals(D), i the absence (negative contra) and presence of AP Taakage of proteins was evaluated afer 2¢hof incubation. Daa sre expressed a the mean = sandard deviation ($0) of hee experiments (+) indicates statsialy sient Ailerene (p = 0.05 porapslosis cells, in comparison with the negative control (Fig. 5). Lectin ean bind t0 chitin, chitin oligomers, cellulose and other sac charides in cell walls, inhibiting the fungal growth [4S]. Additionally, lectins ean cause oxidative stress and energeti collapse [31] and also ‘enter fungal cells and block enzymes involved in the synthesis of wall, polymers [44]. ‘Klafke etal. (45) reported the antifungal activity of six plant lectins against C porapsllosi, with MIC and ME ranging from 0.97 to > 500 ug/ml. The lectin from Helianthus anni seedlings showed significant Inhibitory effects against C. tropicalis, C. parpsilosis, C. albicans and Pichia membranfacens [46]. The lectin Casul. was active against C. ‘ruse with MIC and MFC of 125 and 250 ug/ml, respectively [38]. The values detected for Apul are within the range of ative concentrations reported in these studies. For Candida isolates, the fungistatic effect of Apu become evident after the eighteenth hour of incubation, and the treatments at sub-in hibitory concentrations didnot affect the yeast growth, in comparison with the negative control (ig, 2C and D), At the MICs, Apu promoted protein leakage from both C albicans (Pig, 2C inset) and C.paraptloss (ig. 3D, insed) cells after 24, in comparison to the negative contol cals, ‘SEM images revealed that damage occurred in almost all Candida cals when compared to the control (ig. 48 and 6). All teated-ells of CC. albicans (Fi. 4F) and C. porapsioss (Fg. 4H) exhibited malforma- tions with elongations and bulges, with the presence of cellular frag- ments. These alterations may have affected the cell growth in both species; in addition, the loss of cellular integrity was reflected in the reduction of viability detected by flow cytometry analysis. A recent study showed that Casul at MIC caused cell wall damage to C. kruse, ‘with the presence of ruptured cells and cellular debris. Additionally, the ‘author also observed incomplete cell division [38] 3.3. Synergism evaluation ‘The results from our investigation of the combinatorial effets of Apul, with standaed drugs are shown in Table 2. Oxacllin and eefta zidime were chosen for tis test according to the resistance profile of the isolates of S. aureus and P. aeruginosa, Fluconazole, an antifungal drug commonly used for the treatment of candidiasis, was selected for assays with C. albicans and C. parapsiosis. For the isolates in which bacteriostatic effets were not detected, a MIC value > 400 g/mL. was considered inthe calculations. The combination of Apul-oxacilin ds played synergism against resistant S. aureus UFPEDA.670 and UFPEDA. 671, revealing that Apul alone was not able to inhibit their growth, and was highly effective in reducing the MlCzp ofthe oxacilin. An additive effect was detected forthe non-resistant S. aureus UFPEDA.02, with the antibiotic potentiating the lectin effet. For the isolate UFPEDA.672, the combination showed an indifferent effect, sinee there was po tentiating of lectin activity but, conversely, the antibiotic action was Impaired, ‘Synergistic action was observed for the combination of Apul. snd ceftazidime against P. aeruginosa isolate UFPEDA.262, and this com. bination also revealed additive action against the non-resistant isolate ‘UFPEDA.416, potentiating the action of lectin (Table 2). Conversely, for . aeruginosa isolate UFPEDA-261, the combinatorial eect could not be calculated since the MIC» for both compounds in combination could rot be determined, indicating the high resistance of this bacterium to both Apu. and ceftazidime. The combination of Apul-fluconazoe ex hibited additive and synergistic properties against C. albicans and C arapslosis, respectively able 2. The synergic elect on C parapsiosis corresponded to a reduction greater than 8-old ofthe MICs for fu conazoe, which is ver interesting. ‘Combination therapy isthe mast commonly recommended treat ‘ment against microbial infections in intensive care units, since not all Control Fig. 4 Scanning cloron mleroscopy of microbial calls after exposure to Apul at the MIC, Conta (a, €, €, 8) and Apul-teated (b, df W) cols of Stphylcoeas eres non-resistant isolate UFPEDA02 (a,b), Sphylcoccs ‘areas oxaelin resistant isolate UFPEDA-672 (yd, Candida albicans (, 1 and Candida parapets, b) ean be observed S. ures non-resistant ells showed almost no difference between the conto and retmen, bt some tented el appear not to have completed cll division, ar inicated by arrows (0). The resistant iste 672 presented promineaceson cel srfce weithowt ateration ‘of morphology (2, Treated albicans (9 and parepaan(h) cll exhibited malformations with elongation (aro) and bulges (erohed), nd presence pathogens are sensitive to monotherapy [47]. The use of natural pro- ‘ducts and synthetic antibiotics combined has increased for strategic ‘control of resistant microorganisms [18). A study developed by Kne- zevie et al. [49] using essential oils from Buealyptus camaldulenis in ‘combination withthe anibiotie polymixin B showed synergistic effects ‘against some multi-drug resistant isolates of Acinetobacter Baur Blesson etl. [50] demonstrated a strong synergistic activity of aqueo leaf extract from Colocasia esculema in combination with gentamicin Mera Paogns 1242018) 152-162 against methicilinresistant S. aureus (MIRSA), Curcumin, a natural polyphenolic flavonoid isolated from the shizeme of Curcuma longa, reduced the MIG of several antibiotics, including exacilia, ampicilin, ciprofloxacin and norfloxacin against MRSA [51]. The synergistic ef fects involving alkaloids and fluconazole against resistant Candida species have also been reported {52,53} 34, Antbioiimactvity Biofilm formation is involved in several types of human infections, such as urinary, endocarditis, eystie Abross, dental plaque and many ‘other disorders [5!). The biofilm development phases ince attach ‘ment of eels toa surface, formation of microcolonies, development of| nascent biofilm, differentiation of structured mature biofilm, and dis persil of mature biofilm (55). The eradication of mature biofilms with conventional antibiotic therapy in vivo is almost impessible due to the high drug concentration necessary for effectiveness, increasing the toxicity and side effects on human health [55,56] n this way, the first stage of biofilm development, which involves the adhesion and for ‘ation of microcolonies, isthe most attractive to control biofilms sociated infections [9,57]. In this sense, we investigated the eapacity of ‘Apu. o impale bioflm development by the microorganisms for which \we had determined the MIC. (S. aureus non-resistant UFPEDA.02 and ‘oxacilin-resistant UFPEDA.672 isolates, Cableans and C. raps. Growth and biofllm formation by S. aureus UFPEDA.02 cells were ignificantly (p < 0.05) reduced by 32-85% and 50-60%, respectively, after ApuL treatment in a range of 1.56-50 g/mL, in comparison to non-treated cells (Pig. 6A). Apul.(12.5-400 pg/ml.) and the antibiotic agent (0.031 yg/m) did not present any inhibitory effect against bio film formation by S. aureus UFPEDA-672 (Fig. 6B). Significant inhibi tion of biofilm development was observed for C. albicans at all con centrations evaluated (Pi. 6C). This inhibition was up 9 70%. On the other hand, for C.paropilss, this activity wae minimal, not exceeding 256 inhibition ater ApuL treatment (Fig. 6D). Fluconazole (0.25 yg/ ‘mL) presented a strong (76%) capacity to block biofilm formation by C albicans but, at 128 ug/ml, presented only weak bility to prevent bioiim formation by C. parapllosts, similar to tha which as observed for the lectin, The anibioflm activity of lectins may involve the ability fof these molecules to alter cell viability, interact with constituents present in the exopolymeric matrix of the biofilm, interrupting poly merization, and inhibiting quorum-sensing signals (9,98 Other natural products have already been tested and presented the capacity of blocking biofilm development [ 1. The lectin Casal Impaired biofilm formation by S. aureus (non-resistant and oxacilin resistant isolates) and S. sapropitices after 24h of treatment [35] Jayanth et al. [59] described thatthe lectin from hemolymph of biue swimmer crab Portus pelgicus and is Ag-nanoparticles- conjugated form decreased the quantity of extracellular polymere substances produced by biofilm cells disturbing the biofilm architeeure and in biting the growth of Proteus vulgaris, P. aeruginosa, Enterococcus fo calls and Bacius punils in a dose-dependent manner, 4, Conclusion ‘The present study showed that Apul. presents diferent effects against non-resistant and oxacilin-resistant isolates of S. aureus, as well 1s antifungal activity against Candida. The growth inhibition of 5 aureus, Cabicans, and C. parapsilsis was associated with the imps ‘ment of cell viability. Evaluation of growth curves, protein leakage and tultastrutural changes suggested distinet mechanisms of action by Apu This lectin also exhibited synergistic ation in combination with ‘oxacilin and fluconazole toward oxailin- resistant S. aureus isolate and . parapsloss, respectively. In addtion, anibiofilm effets were de {ected for certain isolates GS. Foren at Mota Paton 1242018) 152-162 Candide albicans Candida parapstosi, 5 xe See a 2eR a UR a a 5 PF nail ~~ - ° a 2a, 3 ay, 7 “haw 32 bow ea4R| af | | . one Jo ro M8 oe ne Fe Aa Fig. 5. How cytometry analysis ofthe cel viability of Candid albicans and Candida paps treated wit Apu a the Mian and their uneated countepans. Cells incubated in absence of etn corresponded othe nqatve control (NC. Iopropyl alcohol 70%, /) was used as postive contol PO. The Ls dot plots can ese (counting bonds are not shot). The hr charts spay the mn orescence i the FL chanel, which coresponds tothe taining by propidiam edie. Data are expres asthe mean = standard deviation (SD) ifeen letters indent significant diferences(p ~ 0.05) between the teats A ‘Staphylococcusaureus(UFPEDAO2) ‘teflon: ifn trson “Stphylococcus urevsUFPEDA-672) 4 -oPEREEEE “~ = “fs om = os ./ “ep %, oo a cuese 7,7) ee 7 Fig. 6, Ambion setivity of Apulagsnst Stpylococeus cures non estantivlte UFPEDA.02 (A) and oxailinsesitant isolate UFPEDA 672 (B), Candida ‘abbas (C) and Ganda paras (D). The cells were treated fr 24h with sb inhibitory concentrations unl the MIC, fo ea late. The bacterial growth and ‘oti formation were determined by measuring opal deat at 600 nm and bythe crystal volt method, respectively. Non teated ells corresponded to the negative contro (-indcate ait difference (p< 0.05) between the eaters and negative conrl ar analyud by Student's ext. The reference drugs were sed a the MIC Se Tale 2, ‘Acknowledgements ‘The authors express their gratitude to the Conselho Nacional de Desenvolvimento Cienifico e Teenoigico (CNPq; 446902/2014-8) for ‘research grants and fellowship (LCBBC, DMAFN, PMGP and THN), the Coordenagéo de Aperfeicoamento de Pessoal de Nivel Superior (CAPES; AUXPE 1454/2013) and the Fundaedo de Amparo & Ciénca e Tecnologia do Estado de Pernambuco (FACEPE; APQ-0108-2.08/14; APQ-0661- 2.08/15) for financial support. GRSF would like to thank CNPq for ‘graduate scholarship. NDLS and MCM would like to thank CAPES for ‘postdoctoral scholarships. The authors als thank Carls Eduardo Sales da Silva for technical assistance. Appendix A. Supplementary data Supplementary data related to this article ean be found at hitps// oi.org/10.1016/} micpath 2018.08.027. 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