Chromosomal Integration of the Klebsiella pneumoniae carbapenemase gene (blaKPC) in Klebsiella Species: Elusive but not Rare

Carbapenemase genes in Enterobacteriaceae are mostly described as being plasmid-associated. However, the genetic context of carbapenemase genes is not always confirmed in epidemiological surveys, and the frequency of their chromosomal integration is therefore unknown. A previously sequenced collection of blaKPC-positive Enterobacteriaceae from a single US institution (2007 2012; n=281 isolates, 182 patients) was analyzed to identify chromosomal insertions of Tn4401, the transposon most frequently harboring blaKPC. Using a combination of short- and long-read sequencing, we confirmed five independent chromosomal integration events from 6/182 (3%) patients, corresponding to 15/281 (5%) isolates. Three patients had isolates identified by peri-rectal screening and three had infections which were all successfully treated. When a single copy of blaKPC was in the chromosome one or both of the phenotypic carbapenemase tests were negative. All chromosomally integrated blaKPC were from Klebsiella spp., predominantly K. pneumoniae clonal group (CG)258, even though these represented only a small proportion of the isolates. Integration occurred via IS15-ΔI mediated transposition of a larger, composite region encompassing Tn4401 at one locus of chromosomal integration, seen in the same strain (K. pneumoniae ST340) in two patients. In summary, we identified five independent chromosomal integrations of blaKPC in a large outbreak, demonstrating that this is not a rare event. blaKPC was more frequently integrated into the chromosome of epidemic CG258 K. pneumoniae lineages (ST11, ST258, ST340), and was more difficult to detect by routine phenotypic methods in this context. The presence of chromosomally integrated blaKPC within successful, globally disseminated K. pneumoniae strains is therefore likely underestimated

Enhanced Klebsiella pneumoniae carbapenemase (KPC) expression from a novel Tn4401 deletion

The Klebsiella pneumoniae carbapenemase gene (blaKPC) is typically located within the mobile transposon Tn4401 Enhanced KPC expression has been associated with deletions in the putative promoter region upstream of blaKPC Illumina sequences from blaKPC-positive clinical isolates from a single institution were mapped to a Tn4401b reference sequence, which carries no deletions. The novel isoform Tn4401h (188bp deletion [lsqb]between istB and blaKPC[rsqb]) was present in 14% (39/281) clinical isolates. MICs for Escherichia coli strains containing plasmids with Tn4401a and Tn4401h were more resistant to meropenem (≥16, ≥16), ertapenem (≥8, 4) and cefepime (≥64, 4) than E. coli strains with Tn4401b (0.5, ≤0.5, ≤1). Quantitative RT-PCR demonstrated that Tn4401a had a 16-fold and Tn4401h a 4-fold increase in blaKPC mRNA levels compared to the reference Tn4401b. A lacZ reporter plasmid was used to test the activity of the promoter regions from the different variants and showed that the Tn4401a and Tn4401h promoter sequences generated higher β-galactosidase activity than the corresponding Tn4401b sequence. Further dissection of the promoter region demonstrated that putative promoter P1 was not functional. Activity of the isolated promoter P2 was greatly enhanced by inclusion of the P1-P2 intervening sequence. These studies indicate that gene expression could be an important consideration in understanding resistance phenotypes predicted by genetic signatures in the context of sequencing-based rapid diagnostics

Enhanced Klebsiella pneumoniae carbapenemase (KPC) expression from a novel Tn4401 deletion

The Klebsiella pneumoniae carbapenemase gene (blaKPC) is typically located within the mobile transposon Tn4401 Enhanced KPC expression has been associated with deletions in the putative promoter region upstream of blaKPC Illumina sequences from blaKPC-positive clinical isolates from a single institution were mapped to a Tn4401b reference sequence, which carries no deletions. The novel isoform Tn4401h (188bp deletion [lsqb]between istB and blaKPC[rsqb]) was present in 14% (39/281) clinical isolates. MICs for Escherichia coli strains containing plasmids with Tn4401a and Tn4401h were more resistant to meropenem (≥16, ≥16), ertapenem (≥8, 4) and cefepime (≥64, 4) than E. coli strains with Tn4401b (0.5, ≤0.5, ≤1). Quantitative RT-PCR demonstrated that Tn4401a had a 16-fold and Tn4401h a 4-fold increase in blaKPC mRNA levels compared to the reference Tn4401b. A lacZ reporter plasmid was used to test the activity of the promoter regions from the different variants and showed that the Tn4401a and Tn4401h promoter sequences generated higher β-galactosidase activity than the corresponding Tn4401b sequence. Further dissection of the promoter region demonstrated that putative promoter P1 was not functional. Activity of the isolated promoter P2 was greatly enhanced by inclusion of the P1-P2 intervening sequence. These studies indicate that gene expression could be an important consideration in understanding resistance phenotypes predicted by genetic signatures in the context of sequencing-based rapid diagnostics

Urinary prostasin in normotensive individuals: correlation with the aldosterone to renin ratio and urinary sodium.

Prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease, activates the epithelial sodium (Na) channel (ENaC), and prostasin is released in extracellular fluids, including urine. Previous data have suggested a direct association between urinary prostasin and the activation of an aldosterone-driven pathway, but a quantitative association has never been demonstrated in normotensive subjects. Similarly, physiological relationships with natriuresis or possible gender- or female hormone-related changes in urinary prostasin concentrations have never been investigated. We measured urinary prostasin by enzyme-linked immunosorbent assay in 43 healthy normotensive subjects of similar age presenting different urinary Na levels and in 15 women during the menstrual cycle and after oral estro-progestinic contraceptive (OC) therapy. Exosomal urinary prostasin was also estimated by western blotting of samples from six healthy subjects twice during the morning. Urinary prostasin presented a wide range of values (from 0.5 to 18.9\u2009nM) without gender differences. It was positively correlated with the aldosterone to renin ratio (ARR) but not with circulating aldosterone or renin individually. Urinary prostasin was directly correlated with U-Na levels (up to 200\u2009nmol Na), whereas it decreased for higher Na concentrations. In women, no significant changes of prostasin concentration were observed during menstrual phases. After OC therapy, prostasin increased (from 2.37\ub11.27 to 4.85\ub15.28\u2009nM), although the increase was not statistically different (P=0.07). Prostasin was detectable in urinary exosomes and displayed a pattern similar to urinary prostasin in relation to urinary Na. In conclusion, urinary prostasin correlates with the ARR, and it is physiologically modulated by natriuresis in normotensive individuals