Isolation of actinomycetes producing bioactive ...

Journal de Mycologie Médicale 15 (2005) 45–51

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SHORT COMMUNICATION

Isolation of actinomycetes producing bioactive substances from water, soil and tree bark samples of the north–east of Algeria Isolement d’actinomycètes producteurs de substances bioactives à partir d’échantillons d’eau, de sol et d’écorces d’arbres du Nord-Est de l’Algérie M. Kitouni a,b,*, A. Boudemagh a,b, L. Oulmi b, S. Reghioua b, F. Boughachiche b, H. Zerizer b, H. Hamdiken b, A. Couble a, D. Mouniee a, A. Boulahrouf b, P. Boiron a a

UMR CNRS 5557, Ecologie Microbienne (Center for Microbial Ecology), Groupe de Recherche “Pathogènes opportunistes et environnement”, Laboratoire de Mycologie, Faculté de Pharmacie, Université Claude Bernard Lyon 1, 8, avenue Rockefeller, 69373 Lyon cedex 8, France b Laboratoire de Microbiologie, Appliquée Université, Mentouri Constantine, Laboratoire de Génie microbiologique et applications, Biopôle Campus, CHAAB-ERSSAS, 25000 Route, Ain-El-Bey Constantine, France Received 8 September 2004; accepted 1 December 2004

KEYWORDS Actinomycetes; Water; Soil; Tree barks; Antimicrobial activity; North–east Algeria

MOTS CLÉS Actinomycètes; Eau; Sol; Ecorces d’arbres; Activité antimicrobienne; Nord–est algérien

Abstract Twenty-five strains of actinomycetes were isolated from samples of water, soil and tree barks collected at two sites located in the north–east of Algeria. Antimicrobial activity was tested using the agar cylinder method against three Gram-positive bacteria, three Gram-negative bacteria, three yeasts and three filamentous fungi. Among the 25 isolates 14 (56%) strains showed an activity against at least one of the test-bacteria studied and two (8%) showed antifungal activity. Ninety-three percent of the active strains were identified by the universal PCR as belonging to the Streptomyces genus and 7% to the Actinomadura genus. © 2005 Elsevier SAS. All rights reserved. Résumé Vingt-cinq souches d’actinomycètes ont été isolées à partir d’échantillons d’eau, de sol et d’écorces d’arbres prélevés de deux sites localisés au Nord–Est de l’Algérie. L’activité antimicrobienne a été effectuée contre trois bactéries à coloration de Gram positives, trois bactéries à coloration de Gram négatives, trois levures et trois champignons filamenteux. Elle a été réalisée par la technique de cylindres d’agar. Parmi les 25 isolats 14 souches (56%) ont montré une activité contre au moins une bactérie-test

* Corresponding author. 1156-5233/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi: 10.1016/j.mycmed.2004.12.005

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M. Kitouni et al. étudiée et 2 (8%) ont montré une activité antifongique. Quatre-vingt treize pour cent des souches actives ont été identifiées par PCR universelle comme appartenant au genre Streptomyces et 7% au genre Actinomadura. © 2005 Elsevier SAS. All rights reserved.

Introduction If the advent of antibiotics represented a new stage in the improvement of the quality and the lasting life, the constant evolution of bacteria antibioresistance represents a major limitation of their use. Indeed, the increasing emergence of multiresistant bacteria throughout the world and the lack of antibiotics to combat such pathogenic agents continue to be the major concern of the medical community [9]. In the United States of America (USA), community-acquired pneumonia infects each year more than 4 million people, of which 20% require hospitalization [9]. The species most frequently isolated during its infections are Streptococcus pneumoniae, Hemophilus influenzae and Moraxella catarrhalis [21]. Thirty-four percent of S. pneumoniae isolated in the USA are resistant to penicillin, 32% of H. influenzae are resistant to ampicillin and 91.5% of M. catarrhalis are resistant to penicillin and 15% to erythromicin [21]. In addition, the vancomycin was the antibiotic of choice for the treatment of the infections caused by methicillin-resistant Staphylococcus aureus, until the appearance of the first resistant strains, initially in Japan [4], then in the USA [3] and in France [16]. To all that is added the resistance of 50% of Enterococcus to vancomycin. These bacteria which until 1990 were regarded as nonpathogenic and in particular Enterococcus faecium which develops among patients under treatment a resistance to several antibiotics [1]. In addition to the bacterial infections, the fungal infections which constituted a problem exclusively relevant to dermatology, have been recognized during the 1990s as a major cause of mortality [13]. According to Pfaller and Wenzel (1992) Candida spp. became the third most usually present isolates found in hemocultures in the USA. Moreover, the death rate due to the infections with Candida, Aspergillus and Fusarium among patients under immunosuppressive chemotherapy or transplant patients is relatively high [13]. Amphotericine B was the most appropriate antifungal agents for the treatment of the majority of the mycoses, nevertheless undesirable side effects are observed during the use of this antifungal agents in particular renal toxicity [13]. Beside the amphotericine B, the antifungals belonging to the

derivatives azoles class are not very toxic, but their efficacies is limited in the treatments of the mycoses infections. In addition, it has been reported that the frequency of multiazole-resistant strains belonging to Candida species other than Candida albicans is increasing [5]. From this report the search for new antibiotics enters a new phase of development. The actinomycetes represent the principal sources of secondary metabolites with anticellular activities, and among-them rare actinomycetes constitutes a potentially important source of novel antibiotics [6]. For that, many laboratories endeavored to diversify the sources of actinomycetes by the use of samples coming from unexplored habitats [8,11,12,14]. Few scientific work were carried out on the presence, the ecological distribution and the diversity of actinomycetes in various ecosystems of Algeria [22] which is a country are spread out over 2,381,741 km2 and which is presented in the form of an immense desert delimited to north by a fringe from 200 to 350 km of width. The aims of this study rest on the isolation and the selection of antibiotics producing actinomycetales strains isolated from various areas of the north–east of Algeria and the molecular identification of these strains.

Materials and methods Samples The samples of soil were collected according to the technique of Pochon and Tardieux [15] from a semiarid soil surrounding the Sebkha of Ain M’lila located at the north–east of Algeria (006°34′E, 036°02′N). Using a large sterile spatula, the first five centimeters of the surface layer of the soil are isolated, then with a small sterile spatula in the layer subjacent (between 5 and 15 cm of depth) 100–150 g of soil were collected and deposited on a sterile aluminum sheet. The large remains are isolated (stones, roots, etc.) and approximately 50 g are placed in a sterile flask and transported as quickly as possible to the laboratory. Water samples, were taken from the tables (basins) of the salt company (ENA Sel Algeria) located at Ain M’lila and from the lakes of Djbel Ouahche mountain located at Constantine, city of the Algerian north-

Isolation of actinomycetes producing bioactive substances –east (006°37′E, 036°22′N) according to the technique of Rodier [19], using a ballasted plunger at which is fixed a sterile polyethylene flask. Water collected is introduced into sterile glass flasks of 250 ml and transported in an icebox at the laboratory. The analyses are carried out at the latest in the 24 h, which follow the samples. Trees barks were collected from Oak (Quercus) and Cedars (Cedrus) of Djbel Ouahche Mountain, by cutting out pieces of barks using a sterile blade. The taken barks are deposited on a sterile aluminum sheet, then recovered in sterile flasks and transported to the laboratory.

Samples treatment One gram of each air-dried soil was mixed with 0.1 g of CaCO3 and incubated at 28 °C for 7 days in an atmosphere saturated with moisture [2]. Water sample was shacked in the presence of phenol (7 mg ml–1) for 10 min and filtered through a membrane millipore (0.45 lm). The spores of actinomycetes are recovered by agitating the membrane with sterile glass balls of 4 mm of diameter in physiological water (NaCl 9 g l–1) [2]. For the barks, 10 g of each sample are crushed and suspended in physiological water, then vigorously mixed for 10 min.

Isolation of actinomycetes Three culture media were used for actinomycetes isolation GLM [yeast extract 3 g l–1 (Merck KGaA, Darmstadt, Germany), malt extract 3 g l–1 (Merck), peptone 5 g l–1 (Merck), glucose 10 g l–1 (Merck), agar 20 g l–1 (Merck, pH 7.2)], Bennett [yeast extract 1 g l–1 (Merck), beef extract 1 g l–1 (Merck), casaminoacids 2 g l–1 (Merck), glucose 10 g l–1 (Merck), agar 15 g l–1 (Merck, pH 7.3)] and glucoseasparagin [di-potassium hydrogen phosphate 0.5 g l–1 (Merck), asparagine 0.5 g l–1 (Prolabo, Paris, France), agar 15 g l–1 (Merck, pH 7.0)]. These media are supplemented with 75 lg ml–1 of amphotericin B to inhibit the development of filamentous fungi and 10 lg ml–1 of polymixin to inhibit Gramnegative bacteria. Inocula consisting of 0.1 ml of dilutions 10–3, 10–4 and 10–5 in the physiological water (NaCl 9 g l–1) of the samples of soil, water and trees barks were spread over the surface of the three culture media. The plates were then incubated at 28 °C. All the plates were observed after 2–4 weeks of incubation at 28 °C. The colonies of actinomycetes were recognized according to their macroscopic characteristic aspect by light microscopy (magnification × 100), then purified, and transferred to GLM slants without antifungal and

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antibacterial, and preserved at 4 °C, and in suspension in GLM in the presence of glycerol to 20% (v/v) at –20 °C.

Antibacterial activity Actinomycetes isolates were grown on Bennett’s and GLM agar plates for 7 days at 28 °C [11]. Agar cylinders (3 mm in diameter) were then taken with hollow punch and deposited on the surface of the Mueller–Hinton media (Merck), which had previously been seeded with each test-bacteria. These last were obtained from the American Type Culture Collection (ATCC) and from the Hospital Juntendo, Tokyo Japan; they are three Gram-positive bacteria (S. aureus ATCC 25923, S. aureus Mu 50 (resistant to vancomycin) and Streptococcus faecalis ATCC 19433) and three Gram-negative bacteria (Escherichia coli ATCC 25922, Proteus vulgaris ATCC 13315 and Pseudomonas aeruginosa ATCC 10145). Plates were kept at 4 °C for 4 h, then incubated at 37 °C for 18–24 h. Inhibition diameters were then measured [12].

Detection of antifungal activity Antifungal activity of actinomycetes isolates was estimated by the agar cylinders method against filamentous fungi obtained from the ATCC and the fungi collection of the Mycology Unit of the Pasteur Institute (UMIP) [Fusarium oxysporum UMIP 625.72, Aspergillus fumigatus UMIP 1082.74 and Aspergillus niger ATCC 16404) and counters the yeasts (C. albicans UMIP 884.65, C. albicans UMIP 48.72 and Candida tropicalis R2 UMIP 1275.81 (resistant to amphotericin B and nystatin)] in the presence of Streptomyces noursei (NRRL B-1714) and Streptomyces nodosus (NRRL B-2371) producing of nystatin and amphotericin A and B, respectively, obtained from Northern Regional Research. The activity was tested in casitone medium [Bacto casitone 9 g l–1 (Difco Laboratories, Sparks, USA), yeast extract 5 g l–1 (Merck), sodium citrate 10 g l–1 (Prolabo), glucose 20 g l–1 (Merck), di-sodium hydrogen phosphate 3.34 g l–1 (Merck), potassium di-hydrogen phosphate 0.54 g l–1 (Merck) and agar 18 g l–1 (Merck)] except for C. tropicalis R2, for which the activity is carried out in YMA medium [yeast nitrogen base 6.5 g l–1 (Difco), asparagin 1.5 g l–1 (Prolabo), glucose 10 g l–1 (Merck) and agar 20 g l–1 (Merck)] [12]. Inhibition diameters were measured after 24 h of incubation at 28 °C for yeasts and after 48 h for filamentous fungi.

Molecular identification of active actinomycetes strains Each strain is identified to the genera or the species level by sequencing of the amplified fragment of

48 the gene 16S rDNA resulting of the polymerase chain reaction (PCR) using the couple of primers 91E (3′ TCAAAK*GAATTGACGGGGGC 5’) and 16S2 (5’ GCCCGGGAACGTATTCAC 3’) [18]. ADN was extracted according to Provost et al. [17]. Into a microtube containing 500 ll of sterile distilled water molecular biology quality is introduced a colony to which 150 ll of extraction suspension (Instagene, Bio-Rad) were added. The mixture was agitated continuously with the vortex for 2 min, then heated for 30 min at 100 °C, then centrifuged for 8 min at 10,000 × g. The supernatant containing the extracted DNA was then recovered in sterile microtubes conserved at –20 °C. Ten microliters of a 1/50 dilution aliquot of the extracted DNA and 15 ll (0.66 pmol ll–1) mixture of the primers couple were introduced into a tube Ready-To-Go™ PCR (Amersham Biosciences) containing Taq DNA polymerase, dNTP, Tris–HCl pH 9, KCl and MgCl2. Amplified reactions were carried out in a thermal cycler (MinicyclerTM MJ Research), after initial denaturation at 94 °C for 10 min followed for 45 cycles of PCR at 94, 55 and 72 °C for 30 s each and finally for 10 min extension period at 72 °C. After amplification, samples were electrophoresed for 2 h at 90 V on 3% agarose gel (Ultra pure, Bio-Rad Laboratories) in TAE 1× buffer containing 0.5 lg ml–1 of ethidium bromide (BET) in the presence of a molecular weights control 100 Base-Pair Ladder (Gibco-BRL). After migration gels were exposed to ultraviolet light (UV) to locate the amplified bands. In the case of the appearance of parasitic bands with the amplified products after electrophoresis, purification is carried out according to the recommendations of the supplier of Kit EZNA (Biofidal).

Results and discussion The choice of the ecosystems studied in this work was carried out for the two following reasons: • Soil sample was collected from the ground surrounding the sebkha of Ain M’lila, the word sebkha is an term of Arab language which is used to design a depression (sometimes a flat zone) occupied by a lake, generally salted; it is a semi-arid soil, of sandy nature, devoid or nearly of vegetation, and a high rate of salinity. Collected water was very salted because it was directly taken from tables (basins) of salts of the Algerian national salt company (ENA Sel Algeria). • Djbel Ouahche mountain, 1200 m of altitude was selected for its richness flora and dense vegetation in addition to its closed lakes (thus very rich

M. Kitouni et al. in humus) and old trees of Oak, tree belonging to the Quercus genus, family of Fagaceae and Cedar which is a tree of the Cedrus genus of the family of Pinaceae. This study was undertaken with an aim of highlighting the presence of actinomycetes in these ecosystems and selecting the strains with antibacterial and antifungal activity. To our knowledge, this is the first study carried out on these ecosystems in Algeria. Isolation of actinomycetes producing of bioactive substances, from extreme ecosystems requires a powerful methods for their selection. Rare actinomycetes are present in small quantity in the various ecosystems and cannot be isolated by the current methods used in microbiology. Their selection must pass by the elimination of the microorganisms, which, by their faculties of invasion, obstruct the growth of actinomycetes. Actinomycetes/microorganisms ratio of a sample increases: • By chemical pretreatment as an example the calcium carbonate and the phenol or physics like the differential centrifugation of the samples [2]. • By the use of certain sources of carbon and nitrogen (starch, chitin, glycerol, casein, arginine, asparagin) making the culture media less favorable to the growth of the bacteria [2]. • Or by the use of antibiotic substances which inhibit the development of the bacteria, fungi and antagonists [10]. Several authors, rightly, consider that the use of antibiotics is an essential precaution in the isolation of actinomycetes. Others combine chemical or physical pretreatment of samples with the addition of antibiotics, but this technique of isolation influences considerably the variation of actinomycetales genera isolated and can be used for a targeted and specific isolation of genera. With this end in view, Larpent and Larpent-Gourgaud (1990) use to isolate the genera Actinomadura, Microbispora, Microtetraspora and Streptosporangium a culture medium supplemented with 50 lg ml–1 of cycloheximide, 50 lg ml–1 of nystatin, 5 lg ml–1 of polymyxin and 1–8 lg ml–1 of penicillin. In order to target the Thermoactinomyces genus they use a medium supplemented with cyclohéximide at a concentration of 50 mg ml–1 and with 25 mg ml–1 of novobiocin, in end to obtain the genera Micromonospora and Nocardia they add 75 mg ml–1 of cycloheximide and 75 mg ml–1 of nystatin in the culture medium used. During our work we adopted this last methodology. To this end we have treated the sample of soil by CaCO3 and water by phenol with 7 mg ml–1 then we have complemented GLM, Bennett’s and glucose–asparagine media with amphotericin B at 75 lg ml–1 and the polymyxin at 10 lg ml–1.

Isolation of actinomycetes producing bioactive substances Results presented in Table 1 reveal an important variation of the number of actinomycetes strains isolated from each sample on the whole of the three selective media. Among the 25 isolated strains of actinomycetes 18 were isolated from telluric samples (soil of sebkha and trees barks), i.e. 72% and only seven from the water samples thus 28%. The presence of a high number of actinomycetes in the soil and trees barks are in agreement with the bibliographical data which let appear the soil as the principal reservoir of actinomycetes [12]. The method used for the screening of antibacterial and antifungal activity of the 25 isolated actinomycetales was a diffusion method on the Mueller–Hinton medium for the bacteria and YMA and casitone media for the fungi. Among the 25 tested isolates, only 14 strains (56%) were active against at least one of the tests-bacteria (Table 1). Isolates 13 and D3 that showed activity against all of the tested bacteria were isolated from the soil of sebkha. Only EC7c isolate presents activity only against S. aureus Mu 50 (Hospital Juntendo) and L2 and 382 against Escherichia coli ATCC 25922, EC3 and EC1 inhibits all the Gram-positive bacteria, 1(3) and EC10 isolates were the unique actino-

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mycete which inhibits only P. vulgaris ATCC 13315 (Table 2). Among the active actinomycetes, only two strains (13 and L′2) presents antifungal activity (Table 3). Isolate 13 inhibit all fungi tested, as for the L′2, did not inhibit A. fumigatus UMIP 1082.74 and F. oxysporum UMIP 625.72 (Table 3). In former studies, it was shown that the isolation rate of actinomycetes with antimicrobial activity is higher than 40% [12] and in others less than 10% [7]. In this study, the rate of isolation of actinomycetes with antibacterial activity was 48% (Table 4) while that of actinomycètes with antifungal activity was 8% (Table 5). The most intense antibacterial activity was obtained with Bennett’s medium (Table 4) what’s confirmed the results obtained by Khattabi et al. (2002). Traditional identification of aerobic actinomycetes requires heavy and tedious techniques. Indeed, actinomycetes constitute a very wide and varied bacterial group whose the membership or not of a given genus can be confused. The study of morphological, physiological and biochemical characters as well as the parietal components, in particular the amino acids and the glucides provides very distinctive specific information. However,

Table 1 Initial screening of actinomycetes strains (culture in agar medium). Tableau 1 Criblage initial des souches d’actinomycètes (culture en milieu gélosé). Samples

Origin

Number of actinomycetes isolates

1 2 3 4 Total

Soil of sebkha (Ain M’lila) Water of sebkha (Ain M’lila) Water of lake (Constantine) Trees Barks (Constantine)

08 04 03 10 25

Number of active actinomycetes against bacteria and/or fungi 05 03 02 04 14

Table 2 Antibacterial activity of isolated actinomycetes. Tableau 2 Activité antibactérienne des actinomycètes isolés. Isolate

Origin

13 A D3 101 1(3) F 382 20r L2 L′2 EC1 EC10 EC3 EC7c

Soil of sebkha Soil of sebkha Soil of sebkha Soil of sebkha Soil of sebkha Water of sebkha Water of sebkha Water of sebkha Water of lake Water of lake Barks of Cedar Barks of Cedar Barks of Cedar Barks of Cedar

E. coli ATCC 25922 + – + + – – + + + – – – –

P. vulgaris ATCC 13315 + + + + – – + – – – + – –

P. aeruginosa ATCC 10145 + – + – – – – – – – – – – –

S. faecalis ATCC 19433 + + + – – + – – + + – + –

S. aureus Mu 50 + + + + – – – + – – + – + +

+: Positive inhibition (diameter of inhibition > 3 mm). –: No inhibition (diameter of inhibition = 3 mm).

S. aureus ATCC 25923 + – + + – + – + – + + – + –

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M. Kitouni et al.

Table 3 Antifungal activity of isolated actinomycetes. Tableau 3 Activité antifongique des actinomycètes isolés. Isolate

Origin

13 L′2 S. noursei NRRL B-1714 S. nodosus NRRL B-2371

C. albicans UMIP 48.72 Soil of sebkha + Water of lake + NRRL +

C. albicans UMIP 884.65 + + +

C. tropicalis R2 UMIP 1275.81 + + –

NRRL

+

–

+

A. fumigatus UMIP 1082.74 + + +

A niger ATCC 16404 + + +

F. oxysporum UMIP 625.72 + + +

+

+: Positive inhibition (diameter of inhibition > 3 mm). –: No inhibition (diameter of inhibition = 3 mm).

Table 4 Percentage of the active strains of actinomycetes cultivated on the GLM and Bennett’s media. Tableau 4 Pourcentage des souches d’actinomycètes actives cultivées sur les milieux GLM et Bennett. Diffusion method Agar cylinders method

Culture medium of active actinomycetes Bennett

Number (%) of active actinomycetes against E. coli ATCC P. vulgaris P. aeruginosa S. faecalis S. aureus S. aureus 25922 ATCC 13315 ATCC 10145 ATCC 19433 Mu 50 ATCC 25923 4 (16) 4 (16) 2 (8) 5 (20) 6 (24) 6 (24)

GLM Total

3 (12) 6 (24)

5 (20) 6 (24)

1 (4) 2 (8)

4 (16) 8 (32)

3 (12) 8 (32)

6(24) 8 (32)

Table 5 Percentage of the active strains of actinomycetes cultivated on the GLM and Bennett’s media. Tableau 5 Pourcentage des souches d’actinomycètes actives cultivées sur les milieux GLM et Bennett. Diffusion method Agar cylinders method

Culture medium of active actinomycetes Bennett

C. albicans UMIP 4872

Number (%) of active actinomycetes against C. albicans C. tropicalis R2 A. fumigatus A niger ATCC F. oxysporum UMIP 127581 UMIP 108274 16404 UMIP 62572 UMIP 88465

1 (4)

1 (4)

1 (4)

1 (4)

1 (4)

1 (4)

GLM Total

2 (8) 2 (8)

2 (8) 2 (8)

2 (8) 2 (8)

1 (4) 1 (4)

2 (8) 2 (8)

1 (4) 1 (4)

Table 6 Taxonomic group of active actinomycetes isolates. Tableau 6 Groupe taxonomique des isolats d’actinomycètes actives. Isolate 13 A D3 101 1(3) F 382 20r L2 L′2 EC1 EC10 EC3 EC7c

Origin Soil of sebkha Soil of sebkha Soil of sebkha Soil of sebkha Soil of sebkha Water of sebkha Water of sebkha Water of sebkha Water of lake Water of lake Barks of Cedar Barks of Cedar Barks of Cedar Barks of Cedar

Taxonomic group Streptomyces spp. Streptomyces spp. Streptomyces spp. Actinomadura spp. Streptomyces spp. Streptomyce spp. Streptomyces spp. Streptomyces spp. Streptomyces spp. Streptomyces spp. Streptomyces spp. Streptomyces spp. Streptomyces spp. Streptomyces spp.

these characters in certain cases fail to classify an isolate in only one genus [20]. For that of, new methods of identification of actinomycetes are necessary. The universal PCR offer a great advantage for the identification of actinomycetes [17]. Results obtained after sequencing and swinging of the sequences obtained in Gen-bank (www.ncbi.nih.gov/blast/blast.cgi) are presented in the Table 6 from which this reveals that 93% of active actinomycetes belong to Streptomyces genus and 7% at Actinomadura genus. These results confirm the abundance of Streptomyces in telluric ecosystems compared to the other genera of actinomycetes. The universal PCR allowed us an identification on the level of genera, but on the level of the species there is an ambiguity that can be explained by the fact that the primers pair used in this work is not specific to actinomycetes on the one hand and allows only the amplification of a fragment of 400 pb of 16S rDNA on the other hand. To

Isolation of actinomycetes producing bioactive substances confirm the membership of an isolate to a givenspecies it is necessary to pass to amplification and the sequencing of all 16S rDNA.

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