System. Appl. Microbiol. 19,381-387 (1996) © Gustav Fischer Verlag· Stuttgart· Jena .

New York

The Value of Cellular Fatty Acid Analysis in the Identification of Oral Yeasts
ELAINE BLIGNAUT1, RENE SENEKAL \ jOHAN L. F. KOCK 2 , ALFIE BOTHA 2 , and jAKOBUS P. j. VAN DER WESTHUIZEN 3
1

2

3

Orpl Microbiology, Faculty of Dentistry, University of Pretoria, 0001 Pretoria, Republic of South Africa Department of Microbiology, and Department of Dermatology, University of the Orange Free State (U.O.F.S.), 9300 Bloemfontein, Republic of South Africa

Received February 9, 1996

Summary
Cellular fatty acids (CFAs) of oral yeast isolates were determined by gas-liquid chromatography using rigidly standardised methods. A total of 67 isolates, previously identified according to conventional biochemical methods, were tested including 21 Candida albicans, 5 Candida glabrata, 1 Candida guilliermondii, 7 Candida krusei, 1 Candida parapsilosis, 2 Candida tropicalis, 2 Rhodotorula spp, 15 Saccharomyces cerevisiae as well as less frequently isolated species like 1 Candida boidinii, 1 Candida catenulata, 3 Candida fabianii, 2 Candida holmii, 1 Candida lusitaniae, 1 Cryptococcus albidus, a black yeast Exophiala jeanselmei, a fungus Lecytophora mutabilis, 1 Pichia anomala and 1 Trichosporon beigelii. Cells were grown to stationary phase at 30°C on a rotary shaker, washed, lyophilized and methylated. The methyl esters were extracted with hexane and gas chromatography performed on a Hewlett Packard model 5830 A gas chromatograph. The relative percentages of palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) were determined and verified by mass spectrometry. A similarity matrix was calculated using the FAMEDATA RELATION PROGRAM. Cluster analysis was subsequently performed and a dendrogram constructed which placed 84% of the isolates in two major groups with group I having a 96% and group II with a 98% similarity. This method clearly distinguished Candida albicans from Candida glabrata, Candida holmii, Candida parapsilosis, Cryptococcus albidus, Exophiala jeanselmei, Lecytophora mutabilis, Rhodotorula glutinis, Rhodotorula rubra and Saccharomyces cerevisaea. In addition this phenotypic characteristic can distinguish Candida holmii from Saccharomyces cerevisiae and Candida glabrata while Exophiala jeanselmei showed a unique fatty acid profile due to a high percentage 18:2. The presence or absence of poly-unsaturated fatty acids like linoleic (18:2) and linolenic (18:3) are major contributing factors in the distinction between the groups. Strains of the same species clustered together, confirming the value of this phenotypic characteristic in the identification of yeasts associated with the oral cavity. Despite the wide variety of oral yeast isolates investigated in this study, CFA analysis did not prove satisfactory as sole identification method in distinguishing between the individual yeast isolates. CFA analysis is a rapid, easily applied and reliable method which can complement other identification methods in yeast characterization. Consideration should be given to the adoption of standardised preparation procedures in order to expand existing information and pool information generated by various researchers for the establishment of a yeast CFA data bank.

Key words: Fatty acid - Yeast species - Oral Yeasts - Differentiation

Introduction The rising incidence of infections caused by yeasts is well documented (Barrett, 1989; Cooper and Silva-Hunter, 1985; Martin et al., 1981; McElroy, 1984; Sandven, 1990; Tawfik et al., 1989). Although not as frequent as
26 System. Appl. Microbiol. Vol. 19/3

bacterial infections, fungal and yeast infections have a fatality rate two to three times that of bacterial infections (McElroy, 1984). This fact necessitates the rapid identification of these organisms in patients. Numerous commer-

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cial test-kits have been developed over the years, some of which are semi- to fully automated (Oblack et aI., 1981) while another can identify yeasts in as little as 4 h of inoculation (Sekhon et aI., 1988; St-Germain and Beauchesne, 1991). Lin and Fung (1987), having reviewed many of these rapid methods for the identification of medically important yeasts, found most of them to be dependent on morphological characteristics to the extent that their rapidity is curtailed. They concluded that future efforts in establishing reliable identification methods should be directed towards enzyme analysis or gas-liquid chromatography (GLC) of cellular components of yeasts. Previous work, comparing conventional and commercial identification systems, performed by the authors of this paper, confirms the view of Lin and Fung (1987) in that the two commercial identification systems used, namely the ATB 32C and Mycotube, respectively proved 71.6% and 52.2% effective in identifying oral yeast isolates when only biochemical characteristics were considered. Possible contributing factors to the unsatisfactory performance of the commercial systems used, are a limited database, the failure of the Mycotube system to inoculate all chambers as well as the fact that many of the yeast isolates are not those commonly found in clinical material. Since its early description by Abel et al. (1963) and Yamakawa and Veta (1964) cellular fatty acid (CFA) analysis by gas-liquid chromatography (GLC) is a widely utilised method for the identification of pathogenic bacteria in medical laboratories. In bacteria the important fatty acids (FAs) are in the range between 12 and 20 carbon atoms. In yeasts however, FAs with 16 (C16) to 18 (C18) carbon atoms predominate (Ratledge, 1994), namely 16:0 (palmitic), 18:0 (stearic), 18:1 (oleic), 18:2 (linoleic) and 18:3 (linolenic). They occur as esters in triacylglycerol, phospholipids, glycolipids or sterols (Boulton and Ratledge, 1983) in membranes and other cytoplasmic organelles such as the mitochondria, plasmalemma, endoplasmic reticulum, nuclei, vacuoles, spores and lipid particles (Hunter and Rose, 1971). In the wine industry the technique is extremely useful in detecting wild yeast contaminants (Tredoux et aI., 1987) or distinguishing between strains within a species (Augustyn et aI., 1990). GLC also became an important method in yeast taxonomy and establishing teleomorph and anamorph relations within yeast genera (Augustyn et aI., 1992; Botha et aI., 1992; Kock et aI., 1985; Viljoen et aI., 1988b; 1989). A clear relation between the utilisation of many carbon compounds, the production of pseudomycelium and the presence of polyunsaturated fatty acids (PUFAs) has been established in some yeasts by Viljoen et al. (1988c). It is generally accepted that cultural conditions and the growth medium can modify the quantity and composition of microbial FAs (Boulton and Ratledge, 1983; Ratledge, 1982) and therefore rigid control should be exercised over these conditions in order to obtain reproducible results. The use of GLC in the identification of medically important yeasts is not widely applied. Gangopadhyay et al. (1979) and Lategan et al. (1981) were the first to utilise this method for the identification of human yeast isolates. Since then it was mainly used by Brondz et al. (1989),

Brondz and Olsen (1990), Marumo and Aoki (1990) and Moss et al. (1982). Each of these workers applied his own technique and yeasts are cultured on different media, for varying lengths of time and at various temperatures resulting in different FA profiles which make comparison difficult. Smit (1991) and Van der Westhuizen (1993), in applying the standardised technique of FA analysis established by Kock (1988), have made some of the worthiest contributions in this field. A clear distinction was made between some basidiomycetous yeast species, e.g. Filobasidiella and Cystofilobasidium, while a relationship was confirmed between Filobasidiella and the anamorph Cryptococcus (Smit, 1991). Van der Westhuizen (1993) could distinguish between other yeast genera of clinical importance namely Cryptococcus, Malassezia, Rhodotorula and Trichosporon which are all characterised by the presence of 18:2 and 18:3 (w 3) FAs as opposed to Geotrichum and Kloeckera which only have 18:2 but not 18:3 (w 3). Candida glabrata in turn, produces neither 18:2 nor 18:3 FAs but only 18:1 FA (Van der Westhuizen, 1993). With this as background it was decided to investigate the use of CFA analysis in the identification of yeasts associated with the oral cavity.

Materials and Methods Isolates. A total of 67 oral yeast isolates was obtained during an epidemiological survey from a remote rural Venda population in the Northern Transvaal, South Africa, as well as from individuals attending the Oral and Dental Hospital, University of Pretoria, Pretoria. The isolates were identified with the Mycotube (Roche Diagnostica) and ATB 32C (Montalieu Vercieu, France) commercial systems as well as according to the conventional biochemical and morphological methods described by Barnett et al. (1990) and Van der Walt and Yarrow (1984). Culture. The procedure by Kock et al. (1985) was followed and is briefly the following: A pre-inoculum was obtained by growing each yeast isolate in 40 ml of medium containing 80 gil glucose (Merck) and 6.7 gil yeast nitrogen base (Difco) in a 250 ml Erlenmeyer flask on a rotary shaker at 160 r. p.m. at 30°C for 16 h. Twenty ml of this inoculum was transferred to 400 ml of the same medium as above in a 11 flask and grown for 48 h or to stationary phase on a rotary shaker at 30°C. Cells were harvested by centrifugation and washed three times in sterile saline solution at 4°C before lyophilization. Preparation of samples. Saponification and methylation was performed in glass test tubes with screw caps according to Kock et al. (1985). This includes adding 5 ml of a 15% KOH in 50% methanol plus 30 !-II of a 6% lauric acid (Sigma) internal standard, in methanol suspension, to 0.12 g of freeze dried cells, boiling the suspension for 1 h and adjusting pH to 2 with cone. HCI (Merck). Methylation was carried out by the additon of 3 ml of a 14% borontrifluoride in methanol solution, followed by flushing with nitrogen gas and heating in a boiling water bath in sealed tubes for 15 min while shaking. Extraction of the methyl esters was done with hexane. Gas chromatographic analysis. Analysis was performed on a Hewlett-Packard model 5830 A gas chromatograph equipped with dual flame-ionization detectors. A 30 m x 0.75 mm i.d. Supelco Wax 10 capillary column was used under the following conditions: injection temperature 170°C; detector temperature 250°C; initial column temperature 145 °C, then increased by

Fatty Acids in Identification of Oral Yeasts 3°C/min to 225°C; held at 225°C for 10 min and then increased to 240°C at a rate of 3°C/min. The flow rate of the carrier gas (nitrogen) was 3.9 mllmin at a column temperature 160°C; hydrogen flow rate 30 mllmin; and air flow rate 300 mllmin. Relative amounts of given fatty acids were calculated from their respective peak areas. Identification of chromatographic peaks. The 16:0 (palmitic acid), 16:1 (palmitoleic acid), 18:0 (stearic acid), 18:1 (oleic acid), 18:2 (linoleic acid) and 18:3 (linolenic acid) peaks were identified by comparison of their retention times with respect to the retention time of the internal lauric acid standard. The percentage of each of these FAs was calculated from the printout. Verification of results. In order to verify FAs present in all lipid fractions, transesterified samples were analysed by gas chromatography - mass spectrometry (GC - MS). Gas chromatograph - mass spectrometer (GC - MS) analysis was carried out on a Hewlett Packard 5890 A gas chromatograph equipped with a Hewlett Packard 5972 MSD.

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GC conditions: Column: HP-1 30 m fused silica capillary, 0.25 mm i.d. 0.25 11m coating; Carrier gas: He at 14 psi head pressure; Temperature programme: Injector 230°C, initial column temperature 140°C, final column temperature 300 °C, ramp rate 5°C/min--, final time 20 min; Injection: Samples were dissolved in 200 III chloroformlhexane (1:4) and 0.5 III injected with a 50:1 split. Mass spectrometer parameters: EMV 1447, Amu Gain 619, Scan 50 to 650 amu, GC - MS interphase temperature 280°C. The mass spectrometer was aurotuned for El operation with PFTBA for mlz 69, 219 and 502 amu. The percentages of FAs were entered into the FAMEDATA RELATION PROGRAMME (FRP) (Smit, 1991) and correlation calculations performed. The distance matrix was calculated by rendering data below 1% equal to zero. No biasing of the data for a chosen FA was performed (Van der Westhuizen et aI., 1991). After this computation procedure, a cluster diagram was
Computer analysis of FAME (fatty acid methyl esters) data.

Table 1. Percentage cellular fatty acids of oral yeast isolates in Group I No. 1 5 6 8 9 10 11 14 15 16 22 37 41 42 43 46 49 53 60 63 67 38 62 3 21 29 44 61 54 36 35 40 64 48 45 65 39 Name
Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida Candida albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans albicans

16:0 18.3 12.7 15.0 15.1 14.6 14.6 18.8 17.0 15.2 15.9 15.3 16.1 16.6 16.0 16.1 16.3 22.4 15.4 17.1 16.2 18.0 13.2 17.9 18.6 18.4 19.7 13.1 15.6 13.4 8.3 8.4 9.9 12.7 12.7 12.8 16.9 8.3

16:1 12.4 17.9 20.1 15.6 16.8 17.3 16.3 15.4 12.8 22.4 15.4 14.2 13.9 14.7 13.4 15.3 14.6 16.5 17.0 15.8 16.7 17.0 20.5 19.4 7.3 15.3 13.5 12.4 14.7 11.6 14.5 10.7 19.0 12.9 13.7 19.6 13.8

18:0 5.0 2.9 1.6 3.8 3.6 2.9 4.1 4.6 5.2 2.3 3.4 3.6 4.3 4.3 4.3 4.5 6.3 3.2 4.2 4.5 4.0 1.4 2.5 3.7 2.9 3.6 2.3 2.0 2.7 3.1 2.5 3.5 1.9 1.0 1.4 3.1 2.2

18:1 34.6 38.1 32.8 42.3 50.5 46.6 39.2 34.6 39.1 32.2 38.2 38.0 36.6 43.1 36.0 36.0 29.8 31.2 42.8 41.2 34.8 43.3 35.3 34.8 38.0 39.3 41.2 45.1 38.0 57.3 50.0 54.9 49.8 45.0 46.5 45.9 51.3 18:1

18:2 25.6 33.0 26.4 19.1 11.9 15.1 17.7 24.8 24.3 24.0 24.9 25.5 25.2 19.0 27.2 23.8 24.2 28.3 16.3 18.7 23.7 25.0 21.7 20.2 30.0 20.6 19.9 17.1 19.4 14.5 18.4 15.4 11.6 22.6 20.6 14.5 18.1

18:3 4.1 4.4 4.1 4.1 2.6 3.5 4.0 3.7 3.5 3.3 2.7 2.7 3.3 3.0 3.0 4.0 2.8 5.5 2.6 3.3 2.8 5.1 2.2 3.4 3.6 1.8 10.0 7.8 10.1 5.1 6.3 5.6 5.1 5.8 5.0 6.2 18:2

Candida catenulata Candida tropicalis Candida tropicalis Pichia anomala Candida guilliermondii Candida fabianii Candida fabianii Candida fabianii Candida krusei Candida krusei Candida krusei Candida krusei Candida krusei Candida krusei Candida boidinii Candida lusitaniae

16:0 = palmitic acid; 16:1 = palmitoleic acid; 18:0 linoleic acid; 18:3 = linolenic acid

= stearic acid;

= oleic acid;

=

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E. Blignaut et al. considering the percentage 16:0, 16:1, 18:0, 18:1, 18:2 and 18:3 FAs, these yeasts showed a similarity of 96% according to the average linkage method (Fig. 1). Although a variety of strains are found, a consistant feature of the Candida albicans isolates is their high percentage of 18:1 FAs. Candida boidinii, also present in this group, and Lecytophora mutabilis, which fall just outside this group, produced according to our analysis method, no detectable 18:3 FA. Lecytophora and Trichosporon only showed a similarity of 92% with all other isolates in this group (Fig. 1). Group II (Table 2) is represented by all the Saccharomyces cerevisiae and Candida glabrata isolates. This group is characterised by the absence of 18:2 and 18:3 FAs with the exception of one Candida glabrata isolate which produced small quantities of 18:2 FA. Considering the percentage of 16:0,16:1,18:0 and 18:1, these yeasts all clustered into one group with a similarity of 98% (Figl). Candida holmii lies close to this group with a 92% similarity to the Group II isolates (Fig. 1). A further distinguishing characteristic of this group is the high percentage of 16:1 with the two Candida holmii isolates revealing the highest percentages of the entire group namely 60.7% and 62.2% (Table 2). Several isolates were obtained which can be considered to be further related than the isolates in Group I and Group II, according to their FA composition (Table 3). Exophiala jeanselmei was the least related to the other yeast isolates studied. Furthermore, the two Rhodotorula

computed from the similarity matrix obtained, utilising CLUSTAN II on a Sperry 1100 multi processor using the average linkage method which is equivalent to the unweighted pair group method (UPGM) (Sokal and Sneath, 1963).

Results and Discussion
The names designated to all isolates are according to their identification with the conventional methods as described by Barnett et al. (1990) and Van der Walt and Yarrow (1984). FA results obtained in this study are given in Tables 1 to 2. Reproducible results were obtained (standard error < 5%) for all isolates tested. This is in accordance with literature (Kock, 1988). The value of the presence or absence of PUFAs, i.e. linoleic (18:2) and linolenic (18:3) acid in the identification of yeasts of clinical importance has recently been established (Smit, 1991; Van der Westhuizen, 1993). Consequently the emphasis will be placed on these PUFAs in the identification of the yeast isolates investigated in this study. On the basis of the presence or absence of 18:2 and 18:3 FAs, the yeasts under investigation can be divided into two major groups. Group I (Table 1, Fig. 1), comprising all the strains of Candida albicans, Candida boidinii, Candida

catenulata, Candida fabianii, Candida guillermondii, Candida krusei, Candida lusitaniae, Candida tropicalis and Pichia anomala, contained both 18:2 and 18:3 FAs. When

Table 2. Percentage cellular fatty acids of oral yeast isolates in Group II No.
12 13 18 23 26 27 28 30 31 32 33 51 52 57 58 2 56 50 17 59 34 55

Name Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae Candida glabrata Candida glabrata Candida glabrata Candida glabrata Candida glabrata Candida holmii Candida holmii

16:0 11.1 11.2 9.1 12.2 9.8 10.8 10.2 11.7 9.0 10.8 12.1 11.6 10.3 12.0 10.7 5.3 6.9 4.3 5.4 5.4 10.5 14.7

16:1 42.2 52.8 46.9 45.8 46.4 48.8 48.2 51.8 43.4 47.8 50.1 48.3 45.8 49.3 45.6 50.7 51.3 48.7 52.0 50.3 62.2 60.7

18:0 5.4 4.8 5.7 6.9 5.5 5.3 5.4 5.2 5.6 6.1 6.3 6.0 6.0 5.8 6.0 5.3 7.3 5.5 6.4 5.9 1.4 2.0

18:1 41.2 31.8 38.3 35.1 38.1 34.6 36.2 31.2 42.0 35.3 31.5 34.1 37.9 32.9 38.0 38.8 34.4 41.6 36.3 35.9 25.2 22.6

18:2

18:3

3.1

16:0 = palmitic acid; 16:1 = palmitoleic acid; 18:0 = stearic acid; 18:1 = oleic acid; 18:2 = linoleic acid; 18:3 = linolenic acid

Fatty Acids in Identification of Oral Yeasts
1.

385

43. 14. 41. 46. 67. 15. 22. 37.
&.

C. albicans C. albicans C. albicans albicans albicans C. albicans C. albicans C. albicans C. albicaos C. a1bicans C. a1bicaos C. albicans C. albicans C. albicans C. albicans C. albicans C. albicans C. albicaos C. albicans C. albicans C. albicans C. boidinii C. catenulata C. fabianii C. fabienii C. fabianii C. guimermondii krusei C. krusei C. krusei C. krusei C. krusei C. krusei C. lusitaoi.. C. tropicalis C. tropicelis P. anomala Lecytophora mutebilis Tr. beigelii C. krusei C. perapsiJosis Cr. albidus Rh. rubra Rh. glutinis Exophiela jeanselm8i C. glebrata C. glabrata C. glebrata C. glabrata C. glabrata Sacch. ceravisiae Sacch. cerevisiae Slcch. cerevisiae Secch. cerevisie8 Slcch. cerevisiee sacch. cerevisiae Sacch. carevisie8 sacch. cerevisiae Secch. carevisiee Slcch. carevisile Sicch. cerevisiae Slcch. cerevisiee Slcch. cerevisiee Slcch. cerevisile Slcch. cerevisiee C. holmii C. holmii

C. C.

GROUP I

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Fig. 1. Dendrogram depicting similarities between cellular fatty acid profiles of 67 oral yeast isolates.

386

E. Blignaut et al. Table 3. Percentage cellular fatty acids of oral yeasts not included in Group I or II No. 7 47 66 4 25 19 24 20 Name 16:0 29.0 19.3 13.8 15.3 18.9 13.5 13.5 17.2 16:1 2.0 6.3 25.6 4.0 2.3 1.2 1.4 5.3 18:0 3.4 13.0 1.7 5.7 10.3 8.4 5.6 2.2 18:1 46.6 44.2 25.1 57.0 54.3 69.4 68.8 25.7 18:2 19.0 21.2 27.2 16.4 13.7 7.5 8.6 49.5 18:3 1.7 6.2 1.6 0.5 1.8 2.0

Lecytophora mutabilis Trichosporon beigelii Candida krusei Candida parapsilosis Cryptococcus albidus Rhodotorula rubra Rhodotorula glutinis Exophiala jeanselmei

16:0 = palmitic acid; 16:1 = palmitoleic acid; 18:0 linoleic acid; 18:3 = linolenic acid

= stearic acid; 18:1 = oleic acid; 18:2 =

species showed a similarity of 99.5%. These results are in accordance with the literature (Smit et al., 1988; Van der Westhuizen et al., 1991; Viljoen et al., 1986; 1988a; 1988b; 1988c; 1989). The one Candida krusei isolate (66) which did not cluster with the other Group II isolates, produced twice the average amount of 16:1 and half the amount of 18:1 of FA than the other Candida krusei isolates. With the increase in the variety of yeasts appearing in clinical specimens (Barrett, 1989; Martin et al., 1981; McElroy, 1984; Sandven, 1990), FA analysis as an identification method will be provided with further opportunities to prove its value.

ration procedures in order to expand existing information and pool information generated by various researchers for the establishment of a yeast CFA data bank. References

Conclusions
1. This method clearly distinguished Candida albicans from Candida glabrata, Candida holmii, Candida parapsilosis, Cryptococcus albidus, Exophiala jeanselmei, Lecytophora mutabilis, Rhodotorula glutinis, Rhodotorula rubra and Saccharomyces cerevisiae. In addition, this phenotypic characteristic can distinguish Candida holmii from Saccharomyces cerevisiae and Candida glabrata while Exophiala jeanselmei showed a unique FA profile due to a high percentage 18:2. 2. The presence or absence of PUFAs like 18:2 (linoleic) and 18:3 (linolenic) are major contributing factors in the distinction between the groups. 3. Strains of the same species clustered together, confirming the value of this phenotypic characteristic in the identification of yeasts associated with the oral cavity. 4. Despite the wide variety of oral yeast isolates investigated in this study, CFA analysis however did not prove satisfactory as sole identification method in distinguishing between the individual yeast isolates. When performed together with relatively simple and rapid supplementary test like the germ tube test and cycloheximide sensitivity, the distinguishing performance between species could be increased. 5. CFA analysis is a rapid, easily applied and reliable method which can complement other identification methods in yeast characterization. Consideration should be given to the adoption of standardised prepa-

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Fatty Acids in Identification of Oral Yeasts
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Prof. E. Blignaut, Oral Microbiology, Faculty of Dentistry, P.O. Box 1266, Pretoria, 0001, Republic of South Africa

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