Klebsiella pneumoniae multirresistente: un problema de salud pública. Estudio de su presencia en el Hospital General Universitario de Alicante

Enterobacterias como Klebsiella pneumoniae y E.coli, junto con otros microorganismos no fermentadores como P. aeruginosa o A. baumanii son de gran importancia a nivel clínico, debido a la reciente aparición de cepas productoras de BLEE y carbapenemasas. Klebsiella es una bacteria Gram negativa capaz de provocar infecciones en el ser humano, de echo aparece en pacientes que estén en estado de cuidados intensivos con mayor frecuencia que en pacientes sanos. La producción de BLEE por parte de muchas cepas de Klebsiella ha provocado que éstas sean mucho más resistentes a gran cantidad de antibióticos y sean más difíciles de eliminar en el paciente. Klebsiella pneumoniae tiene mecanismos de resistencia naturales y resistencias adquiridas, éstas últimas dependen de la cepa de Klebsiella. Un ejemplo de resistencia adquirida es la producción de BLEE que se encargan de romper el anillo betalactámico de gran cantidad de antibióticos inactivándolos, como por ejemplo penicilinas, cefalosporinas y monobactámicos.

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-Angstrom crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 Angstrom, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.

Antibiotic therapy for Klebsiella pneumoniae bacteremia: Implications of production of extended-spectrum beta-lactamases

The prevalence of extended-spectrum beta-lactamase (ESBL) production by Klebsiella pneumonia approaches 50% in some countries, with particularly high rates in eastern Europe and Latin America. No randomized trials have ever been performed on treatment of bacteremia due to ESBL-producing organisms; existing data comes only from retrospective, single-institution studies. In a prospective study of 455 consecutive episodes of Klebsiella pneumoniae bacteremia in 12 hospitals in 7 countries, 85 episodes were due to an ESBL-producing organism. Failure to use an antibiotic active against ESBL-producing K. pneumoniae was associated with extremely high mortality. Use of a carbapenem ( primarily imipenem) was associated with a significantly lower 14-day mortality than was use of other antibiotics active in vitro. Multivariate analysis including other predictors of mortality showed that use of a carbapenem during the 5-day period after onset of bacteremia due to an ESBL-producing organism was independently associated with lower mortality. Antibiotic choice is particularly important in seriously ill patients with infections due to ESBL-producing K. pneumoniae.

Capsule and fimbria interaction in Klebsiella pneumoniae

The capsular polysaccharide and type I fimbriae are two of the major surface-located virulence properties associated with the pathogenesis of Klebsiella pneumoniae. The capsule is an elaborate polysaccharide matrix that encases the entire cell surface and provides resistance against many host defense mechanisms. In contrast, type 1 fimbriae are thin adhesive thread-like surface organelles that can extend beyond the capsular matrix and mediate D-mannose-sensitive adhesion to host epithelial cells. These fimbriae are archetypical and consist of a major building block protein (FimA) that comprises the bulk of the organelle and a tip-located adhesin (FimH). It is assumed that the extended major-subunit protein structure permits the FimH adhesin to function independently of the presence of a capsule. In this study, we have employed a defined set of K. pneumoniae capsulated and noncapsulated strains to show that the function of type I fimbriae is actually impeded by the concomitant expression of a polysaccharide capsule. Capsule expression had significant effects on two parameters commonly used to define FimH function, namely, yeast cell agglutination and biofilm formation. Our data suggest that this effect is not due to transcriptional/translational changes in fimbrial gene/protein expression but rather the result of direct physical interference. This was further demonstrated by the fact that we could restore fimbrial function by inhibiting capsule synthesis. It remains to be determined whether the expression of these very different surface components occurs simply via random events of phase variation or in a coordinated manner in response to specific environmental cues.

Mechanism of uptake of the catecholic (7)-α-formamido cephalosporin BRL 41897A by klebsiella pneumoniae

The catecholic cephalosporin BRL 41897 A is resistant to β-lactamases and is taken up by bacteria via the iron transport system. The uptake of this antibiotic in E.coli uses the Fiu and Cir outer membrane proteins, whereas in P. aerugtnosa it enters via the pyochelin transport system. In this thesis mutants of K. pneumoniae resistant to BRL 41897A were isolated using TnphoA mutagenesis and used to study the mechanism of uptake of BRL 41897A by K. pneumoniae. The activity of BRL 41897A towards the parent strain (M10) was increased in iron depleted media, whereas no significant differences in the resistant (KSL) mutants were observed. Three mutants (KSL19, KSL38and KSL59) produced decreased amounts of certain iron-regulated outer membrane proteins. The uptake of 55Fe-BRL 41897A by M10 in iron-deficient medium was higher than in iron-rich medium. This result indicated the involvement of an iron transport system in the uptake of BRL 41897A by K. pneumoniae. Uptake by the KSL mutants in iron-deficient culture was higher than that by M10. This result, supported by analysis of outer membrane and periplasmic proteins of the KSL mutants, indicates that loss of one outer membrane protein can be compensated by over expression of other outer membrane and/or periplasmic proteins. However, the increased uptake of BRL 41897A by the KSL mutants did not reflect increased activity towards these strains, indicating that there are defects in the transport of BRL 41897A resulting in failure to reach the penicillin binding protein target sites in the cytoplasmic membrane. Southern blotting of chromosomal digests and sequencing in one mutant (KSL19) showed that only one copy of TnphoA was inserted into its chromosome. A putative TnphoA inserted gene in KSL19, designated kslA, carrying a signal sequence was identified. Transformation of a fragment containing the kslA gene into KSL19 cells restored the sensitivity to BRL 41897A to that of the parent strain. Data base peptide sequence searches revealed that the kslA gene in the KSL19 has some amino acid homology with the E. coli ExbD protein, which is involved in stabilisation of the TonB protein. 

Correction: Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation

Whole genome sequence of Klebsiella pneumoniae U25, a hypermucoviscous, multidrug resistant, biofilm producing isolate from India

Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniae species from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1, blaSHV-28, aac(6’)1b-cr, catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1, aac(6’)-Ib, aac(3)-IId, sul1,2, blaTEM-1A,1B, blaOXA-9, blaCTX-M-15, blaSHV-11, cmlA1, erm(B), mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniae strains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniae NTUHK2044, a transposase gene InsH of IS5-13 was found inserted.

Antibiotic-resistant Klebsiella pneumoniae and Escherichia coli high-risk clones and an IncFII(k) mosaic plasmid hosting Tn1 (blaTEM-4) in isolates from 1990 to 2004

We describe the genetic background of bla(TEM-4) and the complete sequence of pRYC11::bla(TEM-4), a mosaic plasmid that is highly similar to pKpQIL-like variants, predominant among TEM-4 producers in a Spanish hospital (1990 to 2004), which belong to Klebsiella pneumoniae and Escherichia coli high-risk clones responsible for the current spread of different antibiotic resistance genes. Predominant populations of plasmids and host adapted clonal lineages seem to have greatly contributed to the spread of resistance to extended-spectrum cephalosporins.

Klebsiella pneumoniae sequence type 11 from companion animals bearing ArmA methyltransferase, DHA-1 β-lactamase, and QnrB4

Seven Klebsiella pneumoniae isolates from dogs and cats in Spain were found to be highly resistant to aminoglycosides, and ArmA methyltransferase was responsible for this phenotype. All isolates were typed by multilocus sequence typing (MLST) as ST11, a human epidemic clone reported worldwide and associated with, among others, OXA-48 and NDM carbapenemases. In the seven strains, armA was borne by an IncR plasmid, pB1025, of 50 kb. The isolates were found to coproduce DHA-1 and SHV-11 β-lactamases, as well as the QnrB4 resistance determinant. This first report of the ArmA methyltransferase in pets illustrates their importance as a reservoir for human multidrug-resistant K. pneumoniae.