Identification and minisequencing-based discrimination of SHV beta-lactamases in nosocomial infection-associated Klebsiella pneumoniae in Brisbane, Australia

Extended-spectrum beta-lactamases (ESBLs) are active against oxyimino cephalosporins and monobactams. Twenty-one Klebsiella pneumoniae isolates obtained between 1991 and 1995 at the Princess Alexandra Hospital in Brisbane, Australia, were subject to amplification and sequencing of the SHV beta-lactamase-encoding genes. Thirteen strains were phenotypically ESBL positive. Of these, six strains carried the bla(SHV-2a) gene and seven strains carried the bla(SHV-12) gene. Eight strains were phenotypically ESBL negative. Of these, seven strains carried the non-ESBL bla(SHV-11) gene and one strain carried the non-ESBL bla(SHV-1) gene. There was complete correspondence between the ESBL phenotype and the presence or absence of an ESBL-encoding gene(s). In addition, it was determined that of the 13 ESBL-positive strains, at least 4 carried copies of a non-ESBL-encoding gene in addition to the bla(SHV-2a) or bla(SHV12) gene. A minisequencing-based assay was developed to discriminate the different SHV classes. This technique, termed first-nucleotide change, involves the identification of the base added to a primer in a single-nucleotide extension reaction. The assay targeted polymorphisms at the first bases of codons 238 and 240 and reliably discriminated ESBL-positive strains from ESBL-negative strains and also distinguished strains carrying bla(SHV-2a) from strains carrying bla(SHV-12). In addition, this method was used to demonstrated an association between the relative copy numbers of bla(SHV) genes in individual strains and the levels of antibiotic resistance.

Upper level, C House, Coorparoo, Brisbane

View along North-West elevation upper level.

Iwan, C House, Coorparoo, Brisbane

As seen from pool deck.

The Nest, C House, Coorparoo, Brisbane

Timber deck with built-in seat overlooking greater landscape beyond.

Pool deck, C House, Coorparoo, Brisbane

View back towards house from deck. with Iwan (right) and filter room (left).

The Nest, C House, Coorparoo, Brisbane

Concrete framework for The Nest (North-West elevation), with timber framework yet to be added.

North-West elevation, C House, Cooparoo, Brisbane

Entry stair in foreground.

Pool deck, C House, Coorparoo, Brisbane

As seen from The Nest above

Outdoor room, C House, Coorparoo, Brisbane

As seen from upper level of house.

Lower level interior, C House, Coorparoo, Brisbane

View to landcape beyond from lower level interior.

Pool deck, C House, Coorparoo, Brisbane

View towards house from pool deck. Iwan on right and stone clad filter room on left.

The Nest, C House, Coorparoo, Brisbane

Concrete framework for The Nest (North-west elevation), with timber framing yet to be added.

North-West elevation, C House, Coorparoo, Brisbane

As seen from pool deck, looking towards The Nest. Kitchen skylight above right.

Iwan interior, C House, Coorparoo, Brisbane

View through timber battens from interior of Iwan.