Mitochondrial Telomeres as Molecular Markers for Identification of the Opportunistic Yeast Pathogen Candida parapsilosis

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Mitochondrial Telomeres as Molecular Markers for Identification of the Opportunistic Yeast Pathogen Candida parapsilosis
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    10.1128/JCM.40.4.1283-1289.2002. 2002, 40(4):1283. DOI: J. Clin. Microbiol. Hiroshi FukuharaJozef Nosek, L'ubomír Tomáska, Adriana Rycovská and  parapsilosis Candida  Opportunistic Yeast Pathogen Markers for Identification of the Mitochondrial Telomeres as Molecular http://jcm.asm.org/content/40/4/1283Updated information and services can be found at: These include:  REFERENCES http://jcm.asm.org/content/40/4/1283#ref-list-1at: This article cites 37 articles, 21 of which can be accessed free CONTENT ALERTS  more»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://journals.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders:  http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to:  onM ar  c h 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j   c m. a s m. or  g /  D  ownl   o a d  e d f  r  om  onM ar  c h 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j   c m. a s m. or  g /  D  ownl   o a d  e d f  r  om   J OURNAL OF  C LINICAL   M ICROBIOLOGY , Apr. 2002, p. 1283–1289 Vol. 40, No. 40095-1137/02/$04.00  0 DOI: 10.1128/JCM.40.4.1283–1289.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved. Mitochondrial Telomeres as Molecular Markers for Identification of the Opportunistic Yeast Pathogen  Candida parapsilosis Jozef Nosek, 1 * L’ubomı´r Tomáška, 2  Adriana Rycˇovská, 1 and Hiroshi Fukuhara 3  Departments of Biochemistry 1  and Genetics, 2  Faculty of Natural Sciences, Comenius University, 84215 Bratislava,Slovakia, and Institut Curie, Section de Recherche, Centre Universitaire Paris XI, 91405 Orsay, France 3 Received 25 July 2001/Returned for modification 14 December 2001/Accepted 24 January 2002 Recent studies have demonstrated that a large number of organisms carry linear mitochondrial DNA molecules possessing specialized telomeric structures at their ends. Based on this specific structural feature of linear mitochondrial genomes, we have developed an approach for identification of the opportunistic yeastpathogen  Candida parapsilosis . The strategy for identification of   C. parapsilosis  strains is based on PCR amplification of specific DNA sequences derived from the mitochondrial telomere region. This assay iscomplemented by immunodetection of a protein component of mitochondrial telomeres. The results demon-strate that mitochondrial telomeres represent specific molecular markers with potential applications in yeastdiagnostics and taxonomy. Several yeast species are associated with opportunistic infec-tions of humans and other mammals. Among them, candidosesare of the greatest clinical importance. These mycoses manifestthemselves as localized, invasive or systemic infections that arefrequently associated with immune deficiencies, AIDS, immu-nosuppressive therapy, anticancer treatments, organ transplan-tations, and various invasive medical procedures. They arecaused mainly by  Candida albicans , but many recent clinicalsurveys have illustrated the rising significance of non- C. albi- cans  infections. As a result, there has been an increased inter-est in the biology and taxonomy of   Candida  species srcinallybelieved to be nonpathogenic (9, 14, 36). C. parapsilosis  is a widespread pathogen, accounting for upto 30% of nosocomial fungemias. It is also associated withseptic arthritis, peritonitis, vaginitis, and nail and skin infec-tions. An increasing prevalence of   C. parapsilosis  has also beenobserved in cases of endocarditis, either indicating a selectiveaffinity of this yeast for endocardial tissues or reflecting itspropensity to colonize damaged skin and gain ingress alongintravascular lines (5, 10, 14, 36).Due to differences in susceptibility of non- C. albicans  speciesto antifungal drugs, rapid and accurate species identification isessential for the implementation of appropriate therapy. Meth-ods for identification and classification of clinically important Candida  species based on phenotypic and/or morphologiccharacteristics do not always lead to unambiguous results, andidentification of a pathogen is sometimes difficult. The recentdevelopment of various molecular techniques has brought sig-nificant improvements in yeast diagnostics and strain typing(33). Molecular typing approaches for  C. parapsilosis  includerestriction fragment length polymorphism analysis, DNA fin-gerprinting, protein and tRNA profiling, PCR, and electro-phoretic karyotype analysis (4, 6, 12, 27, 29, 30, 35). Although mitochondrial DNA (mtDNA) is typically por-trayed as a circular molecule, the mitochondrial genomes of many organisms are linear double-stranded DNA molecules(23). Recent analyses of mtDNA in various yeasts revealedthat closely related species differ in the form of the mtDNA. A relatively high occurrence of the linear form of the mitochon-drial genome was found in species of the genera  Pichia ,  Willi- opsis  and  Candida  (8, 21). Among the special molecular fea-tures of linear mitochondrial genomes are telomeres, thestructures present at the ends of a linear DNA molecule. In-spection of linear mitochondrial genomes revealed several dis-tinct types of mitochondrial telomeres. In the yeast  C. parap- silosis , mitochondrial telomeres consist of long arrays of tandem repeats of a 738-bp unit. Detailed analysis revealedthat the mtDNA molecules terminate with an incomplete re-peat unit possessing a 5   single-stranded extension. The ex-treme end of the molecule is specifically recognized by themitochondrial telomere-binding protein (mtTBP) that protectsthe single-stranded overhang from enzymatic degradation (21,24, 34).Molecular diagnostics in clinical microbiology require rapidand highly selective methods for identification of pathogenicmicroorganisms. In general, species-specific procedures takeadvantage of the unique traits of a pathogenic microorganism.Since mitochondrial telomeres represent a unique feature of the linear form of mtDNA, we propose that they may representspecific molecular markers suitable for identification of organ-isms harboring linear mitochondrial genomes. Here we testedthe pathogenic yeast  C. parapsilosis , whose close relatives (e.g., C. albicans  and  C. tropicalis  [32, 37]) possess circular genomesin their mitochondria. Our results demonstrate that the nucle-otide sequence of mitochondrial telomeres and the antigenicproperties of the protein specifically binding to this sequencehave great potential for facilitating the molecular identificationof   C. parapsilosis. MATERIALS AND METHODS Yeast strains.  Yeasts were obtained from the Centraalbureau voor Schimmel-cultures (CBS), Delft, The Netherlands.  C. parapsilosis  SR23 (CBS 7157) and Saccharomyces cerevisiae  W303-1A are laboratory strains from the collection of  * Corresponding author. Mailing address: Department of Biochem-istry, Faculty of Natural Sciences, Comenius University, Mlynská do-lina CH-1, 84215 Bratislava, Slovakia. Phone: 421.2.60296.536. Fax:421.2.60296.452. E-mail: nosek@fns.uniba.sk.1283   onM ar  c h 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j   c m. a s m. or  g /  D  ownl   o a d  e d f  r  om   the Department of Biochemistry, Comenius University, Bratislava, Slovakia.  C. parapsilosis  strains designated MCO and PL were kindly provided by P. F.Lehmann (Medical College of Ohio, Toledo) and S. A. Meyer (Georgia StateUniversity, Atlanta), respectively. Yeast cells were grown on YPD plates (1%[wt/vol] yeast extract [Difco], 1% [wt/vol] Bacto Peptone [Difco], 2% [wt/vol]glucose, 2% [wt/vol] agar) at 28 ° C.  Ampli fi cation by PCR and gel electrophoresis.  Yeast cells (approximately 10 4 )from a single colony grown overnight on a fresh YPD plate were picked with a yellow tip (Gilson pipette) and resuspended in 20   l of 50 mM KCl – 10 mMTris-HCl (pH 9.0) – 0.1% (wt/vol) Triton X-100. The suspension was heated at 95to 100 ° C for 5 to 10 min and then centrifuged brie fl  y (10,000    g   for 5 s) tosuppress condensation. Alternatively, cells were resuspended in 20 mM NaOH(20   l) and incubated for 5 to 10 min at room temperature. PCRs (20-  l  fi nal volume) were performed with 50 mM KCl – 10 mM Tris-HCl (pH 9.0) – 0.1%(wt/vol) Triton X-100 – 1.25 mM MgCl 2 – 0.2 mM each deoxynucleoside triphos-phate – 0.5  M each primer – 2  l of cell lysate – Taq  DNA polymerase (0.5 to 2 Uper reaction mixture). PCR primers (5  -CTTGTGCTGGCGATGGTTCA-3  ,5  -GCTCTCAATCTGTCAATCCT-3  , 5  -TAAATTTATGTATATGTTTGCA TATATCTTA-3  , and 5  -TAGGGATTGATTATTTACCTATATATTATCA-3  ) were designed with the Vector NTI 4.0 software package (InforMax Inc.) andsynthesized by Genset. Reactions were prepared on ice by combining 18   l of premixed reaction components (master mix) and 2  l of cell lysate (see above). Ampli fi cations were started in a preheated DNA Thermal Cycler 480 (Perkin-Elmer Cetus) with the following standard three-step program: 3 min at 95 ° C,followed by 25 cycles of 45 s at 94 ° C, 1 min at 49 ° C, and 30 s at 72 ° C and then5 min at 72 ° C. The samples were separated by agarose gel electrophoresis (1.5%[wt/vol] containing 0.5  g of ethidium bromide per ml) at 5 to 10 V/cm for 45 to60 min in 90 mM Tris-borate buffer. Immunoblotting.  Yeast cells were grown until the late logarithmic phase inYPD medium (1% [wt/vol] yeast extract, 1% [wt/vol] peptone, 2% [wt/vol]glucose), and cells (0.1 ml of the culture) were washed with double-distilled waterand lysed for 5 min at 95 ° C in 0.1 ml of 1   sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis loading buffer (62.5 mM Tris-HCl [pH 6.8],2% [wt/vol] SDS, 5% [vol/vol] 2-mercaptoethanol, 10% [vol/vol] glycerol) asdescribed by Horvath and Riezman (13). Proteins were separated by SDS – 13%(wt/vol) polyacrylamide gel electrophoresis (16). Resolved proteins were trans-ferred to nitrocellulose  fi lters in transfer buffer (25 mM Tris, 192 mM glycine,20% [vol/vol] methanol [pH 8.3]) with a semidry electroblotter system (PantherHEP-1; Owl Scienti fi c, Portsmouth, N.H.) for 60 min at 200 mA. Filters wereblocked for 2 h at room temperature with blocking solution (20 mM Tris-HCl[pH 7.5], 150 mM NaCl, 2% [wt/vol] skim milk [Difco]) and then incubatedovernight at 4 ° C in blocking solution containing anti-mtTBP polyclonal rabbitantibody SE1785 at a 1:200 dilution (24). Membranes were washed four times with rinsing buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% [vol/vol]Tween 20) and once with rinsing buffer lacking Tween 20 and then incubated with the blocking solution containing a 1:3,000 dilution of a goat anti-rabbitimmunoglobulin G-alkaline phosphatase conjugate (Sigma) for 2 h at roomtemperature. Blots were washed as described above and developed by the addi-tion of 0.3 mg of   p -nitroblue tetrazolium chloride (Sigma) per ml and 0.15 mg of 5-bromo-4-chloro-3-indolylphosphate toluidine salt (Sigma) per ml in alkalinephosphatase buffer (100 mM NaHCO 3 , 1 mM MgCl 2  [pH 9.8]) for 5 to 20 minat room temperature. Miscellaneous.  mtDNA was prepared as described by Defontaine et al. (7),digested with the restriction endonucleases  Bgl II,  Hin dIII,  Pvu II, and  Eco RV(New England Biolabs) in accordance with the manufacturer ’ s instructions, andseparated by agarose gel electrophoresis. Total cellular DNA was isolated from5-ml yeast culture samples as described by Phillippsen et al. (26). DNA samplesfor pulsed- fi eld gel electrophoresis were prepared as described previously (22)and separated on a 0.8% (wt/vol) agarose gel in 45 mM Tris – borate – 1 mMEDTA buffer in a Pulsaphor apparatus (LKB) in contour-clamped homogeneouselectric  fi eld con fi guration. The pulse switching program for chromosomal DNA separation involved two steps of linear interpolation, i.e., 60 to 65 s for 2.5 h,followed by 65 to 600 s for 69.5 h, at 100 V and 9 ° C throughout. Reproducibility of data.  All PCR and immunoblot analyses were repeated atleast twice with the same results. RESULTS AND DISCUSSIONMitochondrial telomeres as molecular markers.  The mito-chondrial genomes of many organisms were found to be rep-resented by linear DNA molecules possessing telomeres (23).The structure and nucleotide sequence of mitochondrial telo-meres appear to be species speci fi c and thus may representuseful molecular markers applicable in molecular diagnostics.To develop an approach for identi fi cation of   C. parapsilosis , we designed two pairs of oligonucleotide primers for PCRampli fi cation (see Materials and Methods). The oligonucleo-tides were derived from nucleotide sequences of   C. parapsilosis mitochondrial telomeres (21) (EMBL data library accessionnumbers X76196 and X76197) and the conserved region of thenuclear 18S rRNA gene (accession number M60307) to serveas  C. parapsilosis -speci fi c and yeast-speci fi c primers, respec-tively. The sensitivity of this system is based on a high redun-dancy of both molecular markers in  C. parapsilosis  cells. Theredundancy of mitochondrial telomeres is due to the repeatednature of telomeric sequences and the presence of multiplecopies of the mtDNA. Similarly, the redundancy of the 18SrRNA gene is due to the presence of 100 to 200 copies withinthe nuclear genome. When cell lysates of   C. parapsilosis  (e.g.,type strain CBS 604) were used as the source of templateDNA, PCR resulted in ampli fi cation of two products of 141and 959 bp derived from the mitochondrial telomere and thegene encoding the 18S rRNA, respectively (Fig. 1).  Analysis of non- C. parapsilosis  species.  Having an optimizedprotocol in hand, we were interested in the speci fi city of thisapproach. We selected 114 yeast strains belonging to 83 dif-ferent species (Table 1). This collection contained mainly  Can- dida  species that are considered to be phylogenetically relatedto  C. parapsilosis , including  C. albicans ,  C. dubliniensis ,  C. maltosa ,  C. tropicalis , and  C. sojae  (1, 18, 19). In addition, yeastspecies known to harbor a linear mitochondrial genome (suchas  Williopsis saturnus ,  Pichia pijperii ,  P. jadinii ,  P. philodendra , C. utilis ,  C. salmanticensis , and  C. vartiovaarae ) were added tothe list. Finally, the panel also included 10 strains of   Loddero- myces elongisporus , as this species was found to be the mostclosely related to  C. parapsilosis  phylogenetically (15, 19) andpreviously was even considered to be its teleomorphic form(11, 20). The results of PCR analysis showed that the  C. parap- silosis -speci fi c product was not ampli fi ed in any of these species FIG. 1. Identi fi cation of   C. parapsilosis  strains with mitochondrialtelomere-derived ( C. parapsilosis -speci fi c) and 18S rRNA gene-derived(yeast-speci fi c) primers. PCR ampli fi cation and gel electrophoresis were performed as described in Materials and Methods. Lanes 1 to 14contained samples of strains CBS 7157, CBS 604 T , CBS 1954, CBS2152, CBS 2193, CBS 2195, CBS 2197, CBS 2211, CBS 2215, CBS2916, CBS 5301, CBS 6318, CBS 8050, and CBS 8181. Arrows indicatethe positions of the mitochondrial telomere-speci fi c PCR product (141bp) and the 18S rRNA-speci fi c PCR product (959 bp).1284 NOSEK ET AL. J. C LIN . M ICROBIOL  .   onM ar  c h 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j   c m. a s m. or  g /  D  ownl   o a d  e d f  r  om   TABLE 1. List of yeast species/strains tested for the presence of the  C. parapsilosis  mitochondrial telomere-speci fi c PCR product Species Strain  a  18S rRNA productMitochondrialtelomereproductSpecies Strain  a  18S rRNA productMitochondrialtelomereproduct Candida akabanensis  CBS 7878 T   Candida albicans  CBS 562 NT   Candida albicans  CBS 1949    Candida albicans  CBS 2716    Candida albicans  CBS 5983    Candida albicans  CBS 6431    Candida apicola  CBS 7444    Candida berthetii  CBS 6113    Candida boidinii  CBS 7447    Candida butyri  CBS 6421 T   Candida cantarellii  CBS 4878 T   Candida caseinolytica  CBS 7881    Candida catenulata  CBS 565 T   Candida catenulata  CBS 2014    Candida catenulata  CBS 6174    Candida cellulolytica  CBS 7920 T   Candida diddensiae  CBS 2214 T   Candida dubliniensis  CBS 7987 T   Candida entomaea  CBS 6306 T   Candida ergatensis  CBS 6248 T   Candida ernobii  CBS 1737 T   Candida ethanolica  CBS 8041 T   Candida fabianii  CBS 5481 T   Candida fermentati  CBS 8302    Candida floricola  CBS 7289 T   Candida fluviatilis  CBS 6776 T   Candida friedrichii  CBS 4114 T   Candida fukuyamaensis  CBS 7921 T   Candida glabrata  CBS 138 T   Candida homilentoma  CBS 6312 T   Candida inconspicua  CBS 180 T   Candida insectamans  CBS 6033 T   Candida intermedia  CBS 572 T   Candida ishiwadae  CBS 7401    Candida krissii  CBS 6519 T   Candida magnoliae  CBS 166 T   Candida magnoliae  CBS 2677    Candida magnoliae  CBS 3086    Candida magnoliae  CBS 6201    Candida maltosa  CBS 5611 T   Candida melibiosica  CBS 5814 T   Candida membranifaciens  CBS 6060    Candida nitratophila  CBS 2027 T   Candida norvegica  CBS 2874    Candida oleophila  CBS 8269    Candida ooitensis  CBS 7299 T   Candida oregonensis  CBS 5036 T   Candida ovalis  CBS 7298 T   Candida paludigena  CBS 8005 T   Candida pararugosa  CBS 1010 T   Candida pignaliae  CBS 6071 T   Candida pini  CBS 970 T   Candida pseudolambica  CBS 2063 T   Candida pseudotropicalis  CBS 607 T   Candida quercuum  CBS 6422 T   Candida rhagii  CBS 4237 T   Candida santjacobensis  CBS 8183 T   Candida sake  CBS 159 T   Candida sake  CBS 5093     a  A superscript capital T or NT indicates the type or neotype strain of the species, respectively. Candida salmanticensis  CBS 5121 T   Candida savonica  CBS 6563 T   Candida sequanensis  CBS 8118 T   Candida shehatae  CBS 5813 T   Candida schatavii  CBS 6452 T   Candida silvae  CBS 5498 T   Candida silvanorum  CBS 6274 T   Candida sojae  CBS 7871 T   Candida sonorensis  CBS 6792 T   Candida sorbophila  CBS 7922    Candida  sp. CBS 5927    Candida  sp. CBS 8262    Candida stellimalicola  CBS 7853 T   Candida succiphila  CBS 7297    Candida tenuis  CBS 615 T   Candida tenuis  CBS 2309    Candida tropicalis  CBS 94 T   Candida tropicalis  CBS 643    Candida tropicalis  CBS 2321    Candida tropicalis  CBS 2323    Candida tropicalis  CBS 6719    Candida tropicalis  CBS 6948    Candida tropicalis  CBS 5701    Candida tropicalis  CBS 7923    Candida utilis  CBS 621 T   Candida vaccinii  CBS 7318 T   Candida vartiovaarae  CBS 4289 T   Candida versatilis  CBS 1752 T   Candida vini  CBS 639    Candida vini  CBS 2122    Candida zeylanoides  CBS 619 NT   Clavispora lusitaniae  CBS 6936 T    Kluyveromyces lactis  CBS 2359     Lodderomyces elongisporus  CBS 1946     Lodderomyces elongisporus  CBS 2605 T    Lodderomyces elongisporus  CBS 2606     Lodderomyces elongisporus  CBS 5912     Lodderomyces elongisporus  CBS 6120     Lodderomyces elongisporus  CBS 6180     Lodderomyces elongisporus  CBS 6181     Lodderomyces elongisporus  CBS 6182     Lodderomyces elongisporus  CBS 6298     Lodderomyces elongisporus  CBS 7803     Pichia canadensis  CBS 1992 T    Pichia jadinii  CBS 1600 T    Pichia kluyveri  CBS 7907     Pichia pastoris  CBS 704 T    Pichia philodendri  CBS 6075 T    Pichia pijperi  CBS 2887 T   Saccharomyces cerevisiae  W303-1A     Williopsis saturnus  var.  mrakii CBS 1707 T   Williopsis saturnus  var.  saturnus CBS 5761 T   Williopsis saturnus  var.  suaveolens CBS 255 T   Williopsis saturnus  var.  suaveolens CBS 1670    Yarrowia lipolytica  CBS 599 T   V OL  . 40, 2002 MOLECULAR DIAGNOSTICS OF  CANDIDA PARAPSILOSIS  1285   onM ar  c h 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j   c m. a s m. or  g /  D  ownl   o a d  e d f  r  om   (Table 1). In 18 strains belonging to 12 different species, nei-ther the mitochondrial telomere-speci fi c nor the 18S rRNA-derived PCR product was ampli fi ed. A closer inspection of these cases revealed that these results were due to the presenceof an inhibitor of   Taq  DNA polymerase srcinating from thecell lysates, since the ampli fi cations performed on puri fi edDNA samples yielded the 18S rRNA PCR product (e.g.,  C. apicola ,  C. magnoliae ,  C. salmanticensis , and  C. shehatae ). Al-ternatively, a divergence in the nucleotide sequence of the 18SrRNA (e.g.,  Yarrowia lipolytica ) may be responsible for the lackof a PCR product.These results demonstrate the speci fi city of a mitochondrialtelomere-derived marker since the corresponding PCR prod-ucts could not be generated in samples from non- C. parapsi- losis  species. The high selectivity of this approach is also illus-trated by the ability to discriminate between  L. elongisporus and  C. parapsilosis. Survey of   C. parapsilosis  strains.  Genetic heterogeneity hasbeen reported in  C. parapsilosis  (2, 4, 5, 18, 25, 30). Recently,it has been demonstrated that  C. parapsilosis  isolates can bedivided into three distinct genotype groups (17, 28). The dif-ferences between these groups are profound, and it was sug-gested they may represent distinct species (5, 6, 17, 28). Due tothe genetic heterogeneity mentioned above, it was of interestto determine whether a molecular marker based on mitochon-drial telomeres could allow discrimination among these groups(Table 2).First, we examined 15 different  C. parapsilosis  strains ob-tained from the CBS yeast collection. All of these strains,except CBS 5301, reproducibly yielded both of the PCR prod-ucts described above, indicating positive identi fi cation as  C. parapsilosis  (Fig. 1). To test the hypothesis that the resultobtained with CBS 5301 was caused by its genetic differencefrom other  C. parapsilosis  strains, we analyzed this case in moredetail. To exclude the possibility that the cells used for analysiscontained incidental contamination, we analyzed a new sampleof this strain from the CBS collection and obtained the sameresults. Based on physiologic and morphologic criteria, E. Sla- vikova (Czechoslovak Culture of Yeasts, Chemical Institute of Slovak Academy of Sciences, Bratislava, Slovakia) recognizedthis strain as non- L  -arabinose-utilizing form II of   C. parapsilo- sis . However, identi fi cation by the API 20C kit (Biomerieux,Marcy l ’ Etoile, France) revealed that the biotype of CBS 5301(i.e., 6176171) differs from that typical of   C. parapsilosis  strains(i.e., 6756171) due to the absence of   L  -arabinose assimilationand weak utilization of   D -xylitol. Also, comparison of the elec-trophoretic karyotypes and mtDNA restriction enzyme diges-tion patterns of CBS 5301 and CBS 604 revealed remarkabledifferences in nuclear and mitochondrial genome organization(Fig. 2) corresponding to the absence of mitochondrial te-lomere-derived PCR products in CBS 5301 cells. Thus, accord-ing to the molecular criteria, strain CBS 5301 seems to besubstantially different from  C. parapsilosis  type strain CBS 604and may represent a distinct species.Next, we analyzed clinical isolates belonging to three differ-ent genotype groups of   C. parapsilosis  as de fi ned by Lin et al.(17) and Roy and Meyer (28). PCR ampli fi cation on lysates FIG. 2. Comparison of electrophoretic karyotypes (A) and  Bgl IIrestriction enzyme digestion patterns of mtDNA (B) of strains CBS5301 (lane 1) and CBS 604 (lane 2) (see Materials and Methods).TABLE 2. List of   C. parapsilosis  strains tested for the presence of mitochondrial telomere-speci fi c PCR product. Species Strain  a  18S rRNA productMitochondrialtelomereproduct Candida parapsilosis  CBS 604 T (I)    Candida parapsilosis  CBS 1954    Candida parapsilosis  CBS 2152    Candida parapsilosis  CBS 2193    Candida parapsilosis  CBS 2194    Candida parapsilosis  CBS 2195    Candida parapsilosis  CBS 2197    Candida parapsilosis  CBS 2211    Candida parapsilosis  CBS 2215 (I)    Candida parapsilosis  CBS 2916    Candida parapsilosis  CBS 6318    Candida parapsilosis  CBS 7157 (SR23)    Candida parapsilosis  CBS 8050 (I)    Candida parapsilosis  CBS 8181 (I)    Candida parapsilosis  CBS 5301    Candida parapsilosis  MCO 433 (I)    Candida parapsilosis  MCO 441 (I)    Candida parapsilosis  MCO 448 (III)    Candida parapsilosis  MCO 456 (II)    Candida parapsilosis  MCO 457 (II)    Candida parapsilosis  MCO 462 (II)    Candida parapsilosis  MCO 471 (II)    Candida parapsilosis  MCO 478 (I)    Candida parapsilosis  PL 429 (III)    Candida parapsilosis  PL 448 (III)    Candida parapsilosis  PL 452 (II)     a  A superscript capital T indicates the type strain of the species. I, II, or III isthe group of   C. parapsilosis  as de fi ned by Lin et al. (17) and Roy and Meyer (28). 1286 NOSEK ET AL. J. C LIN . M ICROBIOL  .   onM ar  c h 1  ,2  0 1 4  b  y  g u e s  t  h  t   t   p:  /   /   j   c m. a s m. or  g /  D  ownl   o a d  e d f  r  om 
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