Role of IS Kpn7 and Deletions in bla _KPC Gene Expression

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>The carbapenemase-encoding<jats:italic>bla</jats:italic><jats:sub>KPC</jats:sub>gene, which is rapidly spreading in Gram-negative rods, is located on a Tn<jats:italic>3</jats:italic>-based transposon, Tn<jats:italic>4401</jats:italic>, which carries a polymorphic region giving rise to five isoforms (<jats:italic>a</jats:italic>,<jats:italic>b</jats:italic>,<jats:italic>c</jats:italic>,<jats:italic>d</jats:italic>, and<jats:italic>e</jats:italic>) that is located immediately upstream of the<jats:italic>bla</jats:italic><jats:sub>KPC</jats:sub>gene and thus likely involved in its expression. Using 5′ rapid amplification of cDNA ends (5′RACE), we identified three potential promoter sequences (P1, P2, and P3) upstream of the<jats:italic>bla</jats:italic><jats:sub>KPC</jats:sub>gene, of which only P1 (absent from isoforms<jats:italic>c</jats:italic>and<jats:italic>d</jats:italic>) and P2 (present in all isoforms, with a −35 box located inside the right inverted repeat of IS<jats:italic>Kpn7</jats:italic>) were shown to be true promoters involved in expression. One representative of each different promoter combination of Tn<jats:italic>4401</jats:italic>, i.e., P2 alone (isoform<jats:italic>c</jats:italic>), P1-P2 (isoform<jats:italic>a</jats:italic>), and P1-P2-P3 (isoform<jats:italic>b</jats:italic>), was cloned into an<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Escherichia coli</jats:named-content>plasmid vector. Using reverse transcription-PCR (RT-PCR), the highest level of expression was obtained with isoform<jats:italic>a</jats:italic>(P1 and P2), which is also the most commonly encountered form in enterobacterial clinical isolates, followed by isoforms<jats:italic>b</jats:italic>(P1, P2, and P3) and<jats:italic>c</jats:italic>(P2 only). These differences in expression led to slight differences in MIC values of carbapenems.<jats:italic>In silico</jats:italic>analysis of the DNA sequence of isoform<jats:italic>b</jats:italic>revealed a stem-loop structure that is likely responsible for strong stops observed in 5′RACE experiments and for decreased expression compared to that with isoform<jats:italic>a</jats:italic>(P1 and P2). In addition, such structures could also be at the origin for the deletions observed in isoforms<jats:italic>a</jats:italic>and<jats:italic>c</jats:italic>. Taken together, these results indicate that the P1 and P2 promoters both contribute to the expression of the<jats:italic>bla</jats:italic><jats:sub>KPC</jats:sub>gene and that the construct with the highest level of expression is that possessing isoform<jats:italic>a</jats:italic>, which is also the most commonly encountered form in clinical isolates.</jats:p>

Comparison of a Novel, Rapid Chromogenic Biochemical Assay, the Carba NP Test, with the Modified Hodge Test for Detection of Carbapenemase-Producing Gram-Negative Bacilli

“…This is likely due to zinc supplementation of the CNP (7). While the CNP detected all carbapenemase-producing GNB in our study, we noticed that color changes were slower for VIM-positive isolates and isolates with low carbapenem MICs (the KPC P. stuartii and OXA-48 K. pneumoniae reference isolates), possibly secondary to inherent differences in hydrolytic activities of VIM and OXA-48 versus KPC and NDM carbapenemases (24)(25)(26) and/or lower expression of carbapenemases in the KPC P. stuartii isolate with low carbapenem MICs (27). The blank tubes included in each run were helpful in this respect; true-positive isolates should exhibit a yellow/orange color, which is more obvious in comparison to the blank.…”

Global Dissemination of bla KPC into Bacterial Species beyond Klebsiella pneumoniae and In Vitro Susceptibility to Ceftazidime-Avibactam and Aztreonam-Avibactam

“…Carbapenem MIC values against KPC-producing bacteria can range from susceptible to fully resistant, with elevated KPC production due to increased bla KPC copy number and/or deletions in the upstream promoter region associated with higher MIC values in some isolates (12,25,26). Production of KPC is often accompanied by loss of either or both of the OmpK35 and OmpK36 porins, which further decreases susceptibility to carbapenems (25,(27)(28)(29).…”

Rapid Induction of High-Level Carbapenem Resistance in Heteroresistant KPC-Producing Klebsiella pneumoniae

“…PCR and sequencing of bla KPC structural region and outer membrane porin genes. We conducted PCR analysis of the Tn4401 regions upstream and downstream of the bla KPC open reading frame with primers based on a report by Naas et al (18) and with primers designed within this study by Primer-BLAST (National Center for Biotechnology Information [NCBI]) ( Table 2). PCR analysis of the coding regions of ompK35 and ompK36 was performed with primers designed by Primer-BLAST.…”