Draft Whole-Genome Sequences of Seven Isolates of Klebsiella pneumoniae from New Zealand Sea Lions

Klebsiella pneumoniae is an opportunistic bacterium causing nosocomial and community-acquired infections. In the 1980s, an invasive form (hypervirulent strain) of K. pneumoniae infection causing primary liver abscesses and septicemia was described (1, 2). Most of the isolates had a hypermucoviscous phenotype (positive string test) and possessed rmpA and rmpA2 genes (1, 3). This hypermucoviscous phenotype has since been reported in animals (4–8).

K. pneumoniae caused mass mortality events in New Zealand sea lion (NZSL) pups in the 2001–2002 and 2002–2003 breeding seasons at Sandy Bay, Subantarctic Enderby Island, New Zealand (9). After these mass mortality events, K. pneumoniae became endemic in this population (10).

Approval for the sampling of live animals was obtained from the New Zealand Department of Conservation Animal Ethics Committee (approval identification numbers AEC52, AEC86, AEC157, AEC158, AEC174, AEC200, and AEC232). K. pneumoniae was isolated from tissues and swabs (Table 1) using MacConkey agar, incubated at 37°C in aerobic conditions. In this study, seven K. pneumoniae isolates from NZSLs were whole-genome sequenced. A NucleoSpin soil kit (Macherey-Nagel, GmbH & Co. KG, Düren, Germany) was used to extract genome-quality DNA from a single colony, which was sent to New Zealand Genomics Limited (Massey Genome Service, Massey University, Palmerston North, New Zealand). A fragment library was prepared using an Illumina TruSeq DNA library preparation kit (version 1) (Illumina, Inc., Scorsby, Victoria, Australia). Paired-end reads (2 × 250 bp) were obtained from a MiSeq instrument (Illumina, Inc., San Diego, CA, USA) and were subject to quality control (Cutadapt, FastQC, and SolexaQA++). The isolates were de novo assembled using SPAdes (version 3.10) (11). The contigs of each isolate produced from SPAdes were annotated by Prokka (version 1.1.2) (12). The sequence type (ST) and serotype of each bacterial isolate were determined using the Bacterial Isolate Genome Sequence Database (BIGSdb) server (http://bigsdb.pasteur.fr/klebsiella/klebsiella.html). Virulence genes were identified by mapping the reads of each isolate along with 1,000-bp flanks on either side of each virulence gene sequence. Genome sizes, numbers of contigs, STs, sources, serotypes, and virulence genes are summarized in Table 1.

Aerobactin, an iron-chelating compound, is suggested to enhance virulence in hypervirulent K. pneumoniae strains (13). However, in this study, two isolates from sea lion pups with fatal K. pneumoniae infections lacked the genes iucD and iutA that code for aerobactin, suggesting that other factors may play roles in their pathogenicity. Another study suggested a relationship between yersiniabactin (encoded by the ybt, irp, and fyu genes) and hypervirulence (14). In our study, ybts and irp2 were found in isolates from both animals with fatal infections and healthy carriers, suggesting that yersiniabactin was not specific for hypervirulence. More studies are needed to clarify the association between chelating compounds and virulence in K. pneumoniae.

One isolate, with a positive string test, did not possess the rmpA and rmpA2 genes considered to play a key role in the hypermucoviscous phenotype (15), suggesting that other factors may contribute to the expression of the hypermucoviscous phenotype (16).

This is the first report of draft whole-genome sequences of K. pneumoniae isolated from NZSLs. The data from this study will provide further information to help understand the genomic relationships of the K. pneumoniae strains that circulate in NZSLs.

This research was supported by the Massey University School of Veterinary Science Research Fund for Postgraduate Students and the Palmerston North Medical Research Foundation (PNMRF).