title: Development of a multiplex-PCR probe system for the proper identification of Klebsiella variicola.
abstract: BACKGROUND
  Klebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species.
  
  
  RESULTS
  This work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates.
  
  
  CONCLUSIONS
  This multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.
authors.affiliation: Departamento de Diagnóstico Epidemiológico, Av. Universidad # 655, Col. Sta. Ma. Ahuacatitlán, C.P. 62100, Cuernavaca, Morelos, Mexico. ulises.garza@insp.mx.
authors.forename: Ulises
authors.initials: U
authors.lastname: Garza-Ramos
authors.affiliation: Departamento de Diagnóstico Epidemiológico, Av. Universidad # 655, Col. Sta. Ma. Ahuacatitlán, C.P. 62100, Cuernavaca, Morelos, Mexico. jsilva@insp.mx.
authors.forename: Jesús
authors.initials: J
authors.lastname: Silva-Sánchez
authors.affiliation: Centro de Ciencias Genómicas (CCG), Universidad Nacional Autónoma de México (UNAM), Cuernavaca, Morelos, Mexico. emartine@ccg.unam.mx.
authors.forename: Esperanza
authors.initials: E
authors.lastname: Martínez-Romero
authors.affiliation: Departamento de Diagnóstico Epidemiológico, Av. Universidad # 655, Col. Sta. Ma. Ahuacatitlán, C.P. 62100, Cuernavaca, Morelos, Mexico. jacinta_87@hotmail.com.
authors.forename: Perla
authors.initials: P
authors.lastname: Tinoco
authors.affiliation: Departamento de Diagnóstico Epidemiológico, Av. Universidad # 655, Col. Sta. Ma. Ahuacatitlán, C.P. 62100, Cuernavaca, Morelos, Mexico. mari_pg_11@hotmail.com.
authors.forename: Marisol
authors.initials: M
authors.lastname: Pina-Gonzales
authors.affiliation: Departamento de Diagnóstico Epidemiológico, Av. Universidad # 655, Col. Sta. Ma. Ahuacatitlán, C.P. 62100, Cuernavaca, Morelos, Mexico. humberto.barrios@insp.mx.
authors.forename: Humberto
authors.initials: H
authors.lastname: Barrios
authors.affiliation: Instituto Nacional de Salud Pública (INSP), Centro de Investigación Sobre Enfermedades Infecciosas (CISEI), Departamento de Inmunología, Cuernavaca, México. jmbarnet@insp.mx.
authors.forename: Jesús
authors.initials: J
authors.lastname: Martínez-Barnetche
authors.affiliation: Instituto Nacional de Salud Pública (INSP), Centro de Investigación Sobre Enfermedades Infecciosas (CISEI), Departamento de Inmunología, Cuernavaca, México. regomez@insp.mx.
authors.forename: Rosa Elena
authors.initials: RE
authors.lastname: Gómez-Barreto
authors.affiliation: Instituto Nacional de Salud Pública (INSP), Centro de Investigación Sobre Enfermedades Infecciosas (CISEI), Departamento de Inmunología, Cuernavaca, México. jmtellez@correo.insp.mx.
authors.forename: Juan
authors.initials: J
authors.lastname: Tellez-Sosa
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001431:Bacteriological Techniques / Q000379:methods*; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D024901:Drug Resistance, Multiple, Bacterial; D006801:Humans; D007231:Infant, Newborn; D007709:Klebsiella / Q000235:genetics / Q000302:isolation & purification*; D007710:Klebsiella Infections / Q000175:diagnosis* / Q000382:microbiology; D008826:Microbial Sensitivity Tests; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D017385:Sequence Homology
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit