Draft Genome Sequence of the First Hypermucoviscous Klebsiella variicola Clinical Isolate

GENOME ANNOUNCEMENT

In the last decade, a new hypervirulent (hypermucoviscous) variant of Klebsiella pneumoniae has been described (1). Most isolates of hypervirulent K. pneumoniae are very susceptible to antimicrobials (except ampicillin). However, a multidrug-resistant and hypervirulent variant of K. pneumoniae has also been described as the next “superbug” (2). On the other hand, Klebsiella variicola is a Gram-negative rod of the Enterobacteriaceae family; it was described as a new bacterial species in 2004 (3). Currently, K. variicola is known to be an endophyte of plants (3, 4), a symbiont in insects (5), and a pathogen in humans (3). A susceptible and multiresistant phenotype of K. variicola has been identified, corresponding to an extended spectrum β-lactamase (ESBL)–producing K. variicola, encoding the SHV-type and CTX-M-15 genes (6, 7).

It is difficult to distinguish K. variicola from K. pneumoniae biochemically as bacterial species. Therefore, it is necessary to use molecular tools such as the rpoB analysis. Accordingly, our teamwork developed a multiplex PCR assay for the proper differentiation of these sister bacteria (7). Using this molecular tool, a screening for antibiotic-susceptible and multiresistant K. pneumoniae clinical isolates was carried out in several Mexican hospitals (7). As a result, the susceptible K. variicola clinical isolate 8917 was identified. This isolate was obtained from the sputum of a 76-year-old man at the Hospital Regional Centenário de la Revolución Mexicana in Morelos, Mexico, in 2011. This isolate was initially identified as a susceptible (except to ampicillin) K. pneumoniae isolate using a MicrosScan Walkaway system (Dade Behring, West Sacramento, CA, USA). Subsequently, it was identified as K. variicola using the M-PCR-1; this was confirmed by the phylogeny analysis of the rpoB gene (7). The hypermucoviscous phenotype of Klebsiella variicola isolate 8917 was determined using the semiquantitative string test (8) and then was considered for whole-genome sequencing.

A total genomic sample of K. variicola isolate 8917 was extracted and purified using the DNeasy kit (Qiagen, Germany). The whole-genome sequence was generated using pyrosequencing on the 454 Roche FLX Titanium platform. The sequence data totaled 250,217 reads, with a range in length of 30 to 953 bp. Reads longer than 500 bp were used for de novo assembly with the CLC Genomics Workbench version 4.0 (CLC bio). In total, 103 contigs with an N₅₀ of 257,189 bp were obtained. The estimated genome size was 5,686,491 bp with a 20× coverage, and 99.93% of the bp were above Q40. Gene prediction and annotation were carried out using the bioinformatic MicroScope platform (9). A total of 5,621 coding DNA sequences and 65 tRNA genes were determined. The BLAST searching analysis of the magA, rmpA, and rmpA2 genes described in hypervirulent K. pneumoniae turned out to be negative on the hypermucoviscous K. variicola 8917 genome. However, the following virulence-associated determinants were positive with different amino acid identities: uge (99.10%), ureA (100%), wabG (99.47%), iroN (64.9%), iutA (72.9%), kfuABC (>98.6%), mceG (53.1%), mrkABCDFHIJ (>86.8%), and entB (99.6) and a nucleotide identity of 100% with wzc-932 (serotype). Further analyses are required to identify the genes involved in the hypermucoviscous phenotype on K. variicola clinical isolate 8917.