Dissemination of the metallo-\(\beta\)-lactamase gene blaIMP-4 in Victoria, Australia


Conclusions


Methods

As part of ongoing surveillance of carbapenemase producing Enterobacteriaceae (CPE), bacterial isolates were sent to the Microbiological Diagnostic Unit (MDU) from hospitals statewide in Victoria, Australia.  After culture on non-selective media, single colonies were picked and DNA extracted using MoBio powersoil kits.  PCR of 587 bp of the blaIMP-4 gene was performed using primers IMP-A 5'-GAA GGY GTT TAT GTT CAT AC-3' and IMP-B 5'-GTA MGT TTC AAG AGT GAT GC-3'.   MALDI-ToF (Biomireux) was used for species identification. 
Whole genome sequencing was performed on DNA extractions using Illumina Nextseq 75 bp paired-end sequencing with Nextera XT libararies.   Overlapping paired-ends were first merged using FLASH Magoc 2011and then the overlappend single end reads were combined with the paired-end reads and  de novo assembled using SPAdes v3.8 Bankevich 2012.  Assembled contig queries were compared against the 741 bp blaIMP-4 (NCBI accession AF244145) subject sequence using BLAST Altschul 1990, as implemented in Abricate.   In silico species identification from the reads and the contigs was performed using Kraken 0.10.5-beta Wood 2014.  Alignment free phylogenetic analysis was performed using Andi v0.1 Haubold 2014.  Species nominations were confirmed based on consensus between MALDI-ToF, Kraken and Andi.   

The context of the blaIMP4 gene was determined through visualisation of the blaIMP-4-containing contig in Bandage Wick 2015.  

Reads for all isolates with blaIMP4 positive by abricate were mapped to the pEL1573 plasmid Partridge 2012 using Snippy.  The ML phylogeny was recovered from the core SNP alignment using FastTree v2.1.8 Price 2010.  From the phylogeny it was clear that there were four distinct groups (Fig 1).  To determine which of the groups most closely the BAM files were viewed in artemis.  In the clade containing the reference, most of the plasmid was covered by reads and the class I integron was mapped to completely at the 5' but not at the 3'.  We de novo assembled isolate 2016-21422 (Klebsiella pneumoniae) has a depth of ( yield of 470721823/5600000 giving 84x).  Reads trimmed of low quality and adapter sequences using Trimmomatic.  Then 300 bp max overlap using FLASH. 50% of reads overlapped.  Single end reads are up to 300 bp.   Assembled using SPAdes in shovill --outdir shovill --force --R1 ../../../pEL1573/nullarbor_pEL1573/2016-21422/R1.fq.gz --R2 ../../../pEL1573/nullarbor_pEL1573/2016-21422/R2.fq.gz --gsize 5600000 --opts '--plasmid --cov-cutoff 10' --minlen 500 
inosine-5 monophosphate (IMP) 

RefSeq:WP_017148586.1"
                     /note="Catalyzes the salvage synthesis of
                     inosine-5'-monophosphate (IMP) and
                     guanosine-5'-monophosphate (GMP) from the purine bases
                     hypoxanthine and guanine, respectively; Derived by
                     automated computational analysis using gene prediction
                     method: Protein Homology."

The contig was annotated using Prokka

Mapping done using the command

Reads ma