Rhinoscleroma is a chronic granulomatous infection of the upper airways caused by the bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. The disease is endemic in tropical and subtropical areas, but its diagnosis remains difficult. As a consequence, and despite available antibiotherapy, some patients evolve advanced stages that can lead to disfiguration, severe respiratory impairment and death by anoxia. Because identification of the etiologic agent is crucial for the definitive diagnosis of the disease, the aim of this study was to develop two simple PCR assays. We took advantage of the fact that all Klebsiella pneumoniae subsp. rhinoscleromatis isolates are (i) of capsular serotype K3; and (ii) belong to a single clone with diagnostic single nucleotide polymorphisms (SNP). The complete sequence of the genomic region comprising the capsular polysaccharide synthesis (cps) gene cluster was determined. Putative functions of the 21 genes identified were consistent with the structure of the K3 antigen. The K3-specific sequence of gene Kr11509 (wzy) was exploited to set up a PCR test, which was positive for 40 K3 strains but negative when assayed on the 76 other Klebsiella capsular types. Further, to discriminate Klebsiella pneumoniae subsp. rhinoscleromatis from other K3 Klebsiella strains, a specific PCR assay was developed based on diagnostic SNPs in the phosphate porin gene phoE. This work provides rapid and simple molecular tools to confirm the diagnostic of rhinoscleroma, which should improve patient care as well as knowledge on the prevalence and epidemiology of rhinoscleroma.
Hart CA, Rao SK (2000) Rhinoscleroma. J Med Microbiol 49: 395–396.
[3]
Canalis RF, Zamboni L (2001) An interpretation of the structural changes responsible for the chronicity of rhinoscleroma. Laryngoscope 111: 1020–1026. doi: 10.1097/00005537-200106000-00016
[4]
Gaafar HA, Bassiouny M, El Mofty M, Badour NM, Nour YA (2000) Experimental intravenous inoculation of Klebsiella rhinoscleromatis bacilli in albino rats: a histopathological and bacteriological study. Acta Otolaryngol 120: 279–285. doi: 10.1080/000164800750001099
[5]
Evrard I, Gruyer X, Desse P, Francois A, Marie JP, et al. (1998) Spheno-ethmoidal rhinoscleroma. Report of a case and review of the literature. Ann Otolaryngol Chir Cervicofac 115: 85–88.
[6]
Brenner DJ, A.G. S, Fanning GR (1972) Differentiation of Enterobacter aerogenes from Klebsiellae by deoxyribonucleic acid reassociation. Int J Syst Bacteriol 22: 193–200. doi: 10.1099/00207713-22-4-193
[7]
？rskov I, ？rskov F (1984) Serotyping of Klebsiella. Methods Microbiol 14: 117–133.
[8]
Brisse S, Fevre C, Passet V, Issenhuth-Jeanjean S, Tournebize R, et al. (2009) Virulent clones of Klebsiella pneumoniae: identification and evolutionary scenario based on genomic and phenotypic characterization. PLoS One 4: e4982. doi: 10.1371/journal.pone.0004982
[9]
Brisse S, Grimont F, Grimont PAD (2006) The genus Klebsiella. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. The prokaryotes A handbook on the Biology of Bacteria. 3rd edition ed. New York: Springer.
[10]
Brisse S, Verhoef J (2001) Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA, gyrA and parC genes sequencing and automated ribotyping. Int J Syst Evol Microbiol 51: 915–924. doi: 10.1099/00207713-51-3-915
[11]
Grimont PAD, Grimont F (2005) Genus Klebsiella. In: Brenner DJ, Krieg NR, Staley JT, editors. Bergey's manual of Systematic Bacteriology. New York: Springer-Verlag. pp. 685–693.
[12]
Abalkhail A, Satti MB, Uthman MA, Al Hilli F, Darwish A, et al. (2007) Rhinoscleroma: a clinicopathological study from the Gulf region. Singapore Med J 48: 148–151.
[13]
Dawlatly EE (1991) Radiological diagnosis of rhinoscleroma–the ‘palatal sign’. J Laryngol Otol 105: 968–970. doi: 10.1017/S0022215100117967
[14]
？rskov I, Fife-Asbury MA (1977) New Klebsiella antigen K 82 and the deletion of five of those previously assigned. Int J Syst Bacteriol 27: 386–387. doi: 10.1099/00207713-27-4-386
[15]
Brisse S, Issenhuth-Jeanjean S, Grimont PA (2004) Molecular serotyping of Klebsiella species isolates by restriction of the amplified capsular antigen gene cluster. J Clin Microbiol 42: 3388–3398. doi: 10.1128/JCM.42.8.3388-3398.2004
Borodovsky M, Mills R, Besemer J, Lomsadze A (2003) Prokaryotic gene prediction using GeneMark and GeneMark.hmm. Curr Protoc Bioinformatics Chapter 4: Unit4 5.
[18]
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402. doi: 10.1093/nar/25.17.3389
[19]
The Universal Protein Resource (UniProt) in 2010. Nucleic Acids Res. 38. (Database issue): pp. D142–148.
[20]
Tatusov RL (2001) Phylogenetic classification of proteins encoded in complete genomes. Available: http://www.ncbi.nlm.nih.gov/COG/. Accessed 2009 Feb 6.
[21]
Frangeul L, Glaser P, Rusniok C, Buchrieser C, Duchaud E, et al. (2004) CAAT-Box, Contigs-Assembly and Annotation Tool-Box for genome sequencing projects. Bioinformatics 20: 790–797. doi: 10.1093/bioinformatics/btg490
[22]
Claros MG, von Heijne G (1994) TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10: 685–686. doi: 10.1093/bioinformatics/10.6.685
[23]
Regue M, Hita B, Pique N, Izquierdo L, Merino S, et al. (2004) A gene, uge, is essential for Klebsiella pneumoniae virulence. Infect Immun 72: 54–61. doi: 10.1128/IAI.72.1.54-61.2004
[24]
Frirdich E, Whitfield C (2005) Characterization of Gla(KP), a UDP-galacturonic acid C4-epimerase from Klebsiella pneumoniae with extended substrate specificity. J Bacteriol 187: 4104–4115. doi: 10.1128/JB.187.12.4104-4115.2005
[25]
Wu KM, Li LH, Yan JJ, Tsao N, Liao TL, et al. (2009) Genome sequencing and comparative analysis of Klebsiella pneumoniae NTUH-K2044, a strain causing liver abscess and meningitis. J Bacteriol 191: 4492–4501. doi: 10.1128/JB.00315-09
[26]
Yeh KM, Chang FY, Fung CP, Lin JC, Siu LK (2006) magA is not a specific virulence gene for Klebsiella pneumoniae strains causing liver abscess but is part of the capsular polysaccharide gene cluster of K. pneumoniae serotype K1. J Med Microbiol 55: 803–804. doi: 10.1099/jmm.0.46368-0
[27]
Arakawa Y, Wacharotayankun R, Nagatsuka T, Ito H, Kato N, et al. (1995) Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid. J Bacteriol 177: 1788–1796.
[28]
Shu HY, Fung CP, Liu YM, Wu KM, Chen YT, et al. (2009) Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates. Microbiology 155: 4170–4183. doi: 10.1099/mic.0.029017-0
[29]
Fang CT, Lai SY, Yi WC, Hsueh PR, Liu KL, et al. (2007) Klebsiella pneumoniae genotype K1: an emerging pathogen that causes septic ocular or central nervous system complications from pyogenic liver abscess. Clin Infect Dis 45: 284–293. doi: 10.1086/519262
[30]
Pan YJ, Fang HC, Yang HC, Lin TL, Hsieh PF, et al. (2008) Capsular polysaccharide synthesis regions in Klebsiella pneumoniae serotype K57 and a new capsular serotype. J Clin Microbiol 46: 2231–2240. doi: 10.1128/JCM.01716-07
[31]
Fouts DE, Tyler HL, DeBoy RT, Daugherty S, Ren Q, et al. (2008) Complete genome sequence of the N2-fixing broad host range endophyte Klebsiella pneumoniae 342 and virulence predictions verified in mice. PLoS Genet 4: e1000141. doi: 10.1371/journal.pgen.1000141
[32]
Rahn A, Drummelsmith J, Whitfield C (1999) Conserved organization in the cps gene clusters for expression of Escherichia coli group 1 K antigens: relationship to the colanic acid biosynthesis locus and the cps genes from Klebsiella pneumoniae. J Bacteriol 181: 2307–2313.
[33]
Hobbs M, Reeves PR (1994) The JUMPstart sequence: a 39 bp element common to several polysaccharide gene clusters. Mol Microbiol 12: 855–856. doi: 10.1111/j.1365-2958.1994.tb01071.x
[34]
Whitfield C (2006) Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu Rev Biochem 75: 39–68. doi: 10.1146/annurev.biochem.75.103004.142545
[35]
Frirdich E, Bouwman C, Vinogradov E, Whitfield C (2005) The role of galacturonic acid in outer membrane stability in Klebsiella pneumoniae. J Biol Chem 280: 27604–27612. doi: 10.1074/jbc.M504987200
[36]
Busch RF (1993) Rhinoscleroma occurring with airway obstruction. Otolaryngol Head Neck Surg 109: 933–936.
[37]
Le Hir P, Marsot-Dupuch K, Bigel P, Elbigourmie TM, Jacquier I, et al. (1996) Rhinoscleroma with orbital extension: CT and MRI. Neuroradiology 38: 175–178. doi: 10.1007/BF00604813
[38]
Kim NR, Han J, Kwon TY (2003) Nasal rhinoscleroma in a nonendemic area: a case report. J Korean Med Sci 18: 455–458.
[39]
Badia L, Lund VJ (2001) A case of rhinoscleroma treated with ciprofloxacin. J Laryngol Otol 115: 220–222. doi: 10.1258/0022215011907028
[40]
Gierczynski R, Jagielski M, Rastawicki W, Kaluzewski S (2007) Multiplex-PCR assay for identification of Klebsiella pneumoniae isolates carrying the cps loci for K1 and K2 capsule biosynthesis. Pol J Microbiol 56: 153–156.
[41]
Turton JF, Baklan H, Siu LK, Kaufmann ME, Pitt TL (2008) Evaluation of a multiplex PCR for detection of serotypes K1, K2 and K5 in Klebsiella sp. and comparison of isolates within these serotypes. FEMS Microbiol Lett 284: 247–252. doi: 10.1111/j.1574-6968.2008.01208.x
[42]
Dutton GG, Parolis H, Joseleau JP, Marais MF (1986) The use of bacteriophage depolymerization in the structural investigation of the capsular polysaccharide from Klebsiella serotype K3. Carbohydr Res 149: 411–423. doi: 10.1016/S0008-6215(00)90061-2
[43]
Robertson GA, Thiruvenkataswamy V, Shilling H, Price EP, Huygens F, et al. (2004) Identification and interrogation of highly informative single nucleotide polymorphism sets defined by bacterial multilocus sequence typing databases. J Med Microbiol 53: 35–45. doi: 10.1099/jmm.0.05365-0
[44]
Whitfield C, Paiment A (2003) Biosynthesis and assembly of Group 1 capsular polysaccharides in Escherichia coli and related extracellular polysaccharides in other bacteria. Carbohydr Res 338: 2491–2502. doi: 10.1016/j.carres.2003.08.010
[45]
Munoz R, Lopez R, de Frutos M, Garcia E (1999) First molecular characterization of a uridine diphosphate galacturonate 4-epimerase: an enzyme required for capsular biosynthesis in Streptococcus pneumoniae type 1. Mol Microbiol 31: 703–713. doi: 10.1046/j.1365-2958.1999.01211.x
