284 Development of a Seven Locus Variable Number Tandem Repeat Scheme for Typing of Klebsiella pneumoniae

Friday, March 19, 2010
Grand Hall (Hyatt Regency Atlanta)
Suzanne Elgohari, MSc, BA , Health Protection Agency, Centre for Infections, London, United Kingdom
Jane F. Turton, PhD, BSc , Health Protection Agency, Centre for Infections, London, United Kingdom
Background: Klebsiella pneumoniae subsp. pneumoniae is an important nosocomial pathogen, which can cause outbreaks. A rapid method of typing, to identify possible routes of transmission would be highly valuable.

Objective: To develop a Variable Number Tandem Repeat scheme to discriminate between clinical isolates of Klebsiella pneumonia and to compare it with the standard pulsed-field gel electrophoresis (PFGE) method.
Methods: The genome sequence of K.pneumoniae MGH 78578 was put through a tandem repeat finder programme which identified 117 loci with tandemly repeated sequences. Eleven were selected and primers designed from the flanking sequences such that all shared a similar melting temperature. A panel of 24 isolates, representing 20 PFGE defined strains was selected for the study. These isolates came from hospitals in the UK and had been referred to the reference laboratory at Colindale, UK. PCR reactions were  carried out at each locus and amplicons separated by agarose gel electrophoresis. Loci at which PCR products were found that varied in size among the isolates were chosen for the scheme. The repeat number for an isolate at each VNTR locus was determined from the size of the amplicon and isolates described by listing these numbers.

Results: Of the eleven VNTR loci screened, seven provided discrimination between isolates. All PFGE defined strains could be distinguished from one another on the basis of their VNTR profiles. Isolates within a PFGE cluster shared the same VNTR profile. The virulent K1 clone of clonal complex (CC) 23, associated with pyogenic liver abscesses, could be unambiguously identified from its characteristic VNTR profile.

Conclusions: A scheme using a combination of seven VNTR loci was as effective as conventional PFGE in distinguishing isolates and provided results more rapidly. Screening for more loci could improve the discriminatory power of the VNTR typing scheme.