Klebsiella pneumoniae Multiresistance Plasmid pMET1: Similarity with the Yersinia pestis Plasmid pCRY and Integrative Conjugative Elements

Dissemination of antimicrobial resistance genes has become an important public health and biodefense threat. Plasmids are important contributors to the rapid acquisition of antibiotic resistance by pathogenic bacteria.The nucleotide sequence of the Klebsiella pneumoniae multiresistance plasmid pMET1 comprises 41,723 bp and includes Tn1331.2, a transposon that carries the bla(TEM-1) gene and a perfect duplication of a 3-kbp region including the aac(6')-Ib, aadA1, and bla(OXA-9) genes. The replication region of pMET1 has been identified. Replication is independent of DNA polymerase I, and the replication region is highly related to that of the cryptic Yersinia pestis 91001 plasmid pCRY. The potential partition region has the general organization known as the parFG locus. The self-transmissible pMET1 plasmid includes a type IV secretion system consisting of proteins that make up the mating pair formation complex (Mpf) and the DNA transfer (Dtr) system. The Mpf is highly related to those in the plasmid pCRY, the mobilizable high-pathogenicity island from E. coli ECOR31 (HPI(ECOR31)), which has been proposed to be an integrative conjugative element (ICE) progenitor of high-pathogenicity islands in other Enterobacteriaceae including Yersinia species, and ICE(Kp1), an ICE found in a K. pneumoniae strain causing primary liver abscess. The Dtr MobB and MobC proteins are highly related to those of pCRY, but the endonuclease is related to that of plasmid pK245 and has no significant homology with the protein of similar function in pCRY. The region upstream of mobB includes the putative oriT and shares 90% identity with the same region in the HPI(ECOR31).The comparative analyses of pMET1 with pCRY, HPI(ECOR31), and ICE(Kp1 )show a very active rate of genetic exchanges between Enterobacteriaceae including Yersinia species, which represents a high public health and biodefense threat due to transfer of multiple resistance genes to pathogenic Yersinia strains

External Guide Sequences Targeting the aac(6′)-Ib mRNA Induce Inhibition of Amikacin Resistance▿

The dissemination of AAC(6′)-I-type acetyltransferases have rendered amikacin and other aminoglycosides all but useless in some parts of the world. Antisense technologies could be an alternative to extend the life of these antibiotics. External guide sequences are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. Thirteen-nucleotide external guide sequences complementary to locations within five regions accessible for interaction with antisense oligonucleotides in the mRNA that encodes AAC(6′)-Ib were analyzed. While small variations in the location targeted by different external guide sequences resulted in big changes in efficiency of binding to native aac(6′)-Ib mRNA, most of them induced high levels of RNase P-mediated cleavage in vitro. Recombinant plasmids coding for selected external guide sequences were introduced into Escherichia coli harboring aac(6′)-Ib, and the transformant strains were tested to determine their resistance to amikacin. The two external guide sequences that showed the strongest binding efficiency to the mRNA in vitro, EGSC3 and EGSA2, interfered with expression of the resistance phenotype at different degrees. Growth curve experiments showed that E. coli cells harboring a plasmid coding for EGSC3, the external guide sequence with the highest mRNA binding affinity in vitro, did not grow for at least 300 min in the presence of 15 μg of amikacin/ml. EGSA2, which had a lower mRNA-binding affinity in vitro than EGSC3, inhibited the expression of amikacin resistance at a lesser level; growth of E. coli harboring a plasmid coding for EGSA2, in the presence of 15 μg of amikacin/ml was undetectable for 200 min but reached an optical density at 600 nm of 0.5 after 5 h of incubation. Our results indicate that the use of external guide sequences could be a viable strategy to preserve the efficacy of amikacin

Vibrio cholerae InV117, a Class 1 Integron Harboring aac(6′)-Ib and bla(CTX-M-2), Is Linked to Transposition Genes

A ca. 150-kbp Vibrio cholerae O1 biotype El Tor plasmid includes bla(CTX-M-2) and a variant of aac(6′)-Ib within InV117, an orf513-bearing class 1 integron. InV117 is linked to a tnp1696 module in which IRl carries an insertion of IS4321R. The complete structure could be a potential mobile element

Inhibition of Cell Division Induced by External Guide Sequences (EGS Technology) Targeting <em>ftsZ</em>

<div><p>EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The <em>ftsZ</em> mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. <em>In vitro</em> reactions showed that EGSs targeting these regions bound <em>ftsZ</em> mRNA and elicited RNase P-mediated cleavage of <em>ftsZ</em> mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region “b” under the control of the T7 promoter was generated. Upon introduction of this plasmid into <em>Escherichia coli</em> BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, <em>E. coli</em> harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting <em>fts</em>Z.</p> </div

Vibrio cholerae InV117, a class 1 integron harboring aac(6')-Ib and blaCTX-M-2, is linked to transposition genes

Fil: Soler Bistué, Alfonso J. C. Fundación Instituto Leloir; Argentina.Fil: Martín, Fernando A. Fundación Instituto Leloir; Argentina.Fil: Petroni, Alejandro. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Servicio de Antimicrobianos; Argentina.Fil: Faccone, Diego. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Servicio de Antimicrobianos; Argentina.Fil: Galas, Marcelo F. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Servicio de Antimicrobianos; Argentina.Fil: Tolmasky, Marcelo E. California State University Fullerton; Estados Unidos.Fil: Zorreguieta, Angeles. Fundación Instituto Leloir; Argentina.A ca. 150-kbp Vibrio cholerae O1 biotype El Tor plasmid includes blaCTX-M-2 and a variant of aac(6 )-Ib within InV117, an orf513-bearing class 1 integron. InV117 is linked to a tnp1696 module in which IRl carries an insertion of IS4321R. The complete structure could be a potential mobile element

Effect of EGSb1 on cell division.

<p>A. The inducer IPTG (1 mM) was added to cultures of <i>E. coli</i> BL21(DE3)(pLysS) harboring the recombinant plasmids pEGSb1 or pEGSb1S when their OD₆₀₀ reached 0.2. The cultures were then incubated for 60 more minutes and cells were examined by microscopy. B. Cells were stained with FM5-95 as described in Materials and Methods and examined by laser scanning confocal microscopy. C. Effect of the expression of EGSb1 or EGSb1S on cell survival. Results are expressed as log₁₀ of mean CFU/ml ± SD. Similar results were observed in 3 independent assays. *: p<0.01.</p

EGS sequences.

<p>EGSAP targets the <i>phoA</i> gene. EGSb1S is the sequence complementary to EGSb1.</p

Secondary structure of <i>ftsZ</i> mRNA and regions targeted by EGSs.

<p>A. Secondary structure of the <i>ftsZ</i> mRNA (nucleotides 105083–106456, accession number NC_000913.2) generated with m-fold software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047690#pone.0047690-Zuker1" target="_blank">[34]</a>. B. Zoom in the two regions selected as targets. Colors of the dots indicate the probability that they exist as single stranded. In decreasing order: red, orange, yellow, green, cyan, blue, violet and black. The sequences targeted by the EGSs are shown shadowed. EGSa2 includes an extra nucleotide at each end with respect to EGSa1, this is indicated by two short lines.</p

Analysis of the activity of EGSs.

<p>A. Binding of EGSs to <i>ftsZ</i> mRNA. The oligoribonuclotides were 5′-end labeled, mixed with different amounts of <i>ftsZ</i> mRNA (from left to right 0, 1, 10, 100, or 500 ng) and analyzed by electrophoresis in 6% native polyacrylamide gel. B. RNase P-mediated cleavage of ³²P-labeled <i>ftsZ</i> mRNA. The RNase P components, M1 RNA and C5 protein were preincubated at 37°C for 15 min, and a mix containing the radiolabeled <i>ftsZ</i> mRNA and the indicated EGS was preincubated 25°C for 2 h. After preincubation both solutions were combined, incubated at 37°C for 90 minutes, and analyzed on 6% denaturing PAGE. The location and size of the expected products of cleavage are shown to the left.</p