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The activities of β-lactams, chloramphenicol, tetracycline, fluoroquinolones and aminoglycosides against Klebsiella pneumoniae C3 (O1:K66, producing porins OmpK35 and OmpK36) and a set of isogenic mutants derived from it lacking the O antigen of lipopolysaccharide (LPS), capsular K antigen, or one or both porins were determined. MICs remained within one dilution step in mutants deficient in antigen O, in capsule or in one of the two porins. No increases in the MICs of aminoglycosides, fluoroquinolones, tetracycline and chloramphenicol were observed for strains deficient in the two porins, but the MICs of ampicillin, cephalothin, cefoxitin, cefotaxime and ceftazidime for this type of mutant increased four- to >256-fold. The highest MICs of β-lactams were obtained in a porin-deficient mutant expressing increased β-lactamase activity. It is concluded that isolated outer membrane alterations in K.pneumoniae are not decisive factors in increasing resistance to antimicrobial agents, but porin loss cooperates with β-lactamase production to increase resistance to β-lactams., This work was funded by grants from Ministerio de Educación y Cultura and Junta de Andalucía, Spain.

Fifteen isolates of Klebsiella pneumoniae producing extended-spectrum β-lactamases (ESBLs) isolated during a nosocomial outbreak were studied. The strains belonged to the same clonal type, as shown by pulsed-field gel electrophoretic analysis of chromosomal DNA. All the isolates were resistant to extended-spectrum cephalosporins, aztreonam, gentamicin, and fluoroquinolones and were susceptible to carbapenems, tobramycin, netilmicin, and amikacin. None of the isolates expressed the OmpK36 porin. Eight isolates, for which the MICs of cefoxitin were ≥64 μg/ml, showed a diminished level or no expression of a 35-kDa porin. The MICs of meropenem, cefotaxime, and cefpirome were three to eight times higher for porin-deficient isolates than for isolates expressing the 35-kDa porin, but the MICs of imipenem increased two times for porin-deficient isolates compared to those for isolates expressing the porin. This MIC increase reverted to a level similar to that for the parental strain when porin-deficient isolates were transformed with the gene coding for the K. pneumoniae porin OmpK36. It is concluded that the high level of resistance to cefoxitin and the increase in the MICs of meropenem, cefotaxime, and cefpirome for the ESBL-producing K. pneumoniae isolates studied are associated with porin deficiency.

FC3-11, a bacteriophage specific for the Klebsiella pneumoniae porin OmpK36, was isolated by its ability to infect Escherichia coli strains expressing the cloned OmpK36 porin. Porin OmpK36 was shown to be the receptor for phage FC3-11 by the observations that K. pneumoniae and E. coli strains that do not express OmpK36 were resistant to phage FC3-11, the purified porin inactivated the phage, and mutants selected for FC3-11 resistance had lost OmpK36. The outer membrane protein OmpK35 was isolated from a K. pneumoniae phage-resistant mutant by using porin isolation methods and was shown to contain an N-terminal sequence typical of enterobacterial porins. Bacteriophage FC3-11, alone or in combination with previously described lipopolysaccharide-specific phages, is a valuable tool to obtain OmpK36-porinless mutants.Key words: Klebsiella pneumoniae, porins, bacteriophage.

Mesophilic Aeromonas spp. strains (serotype O:34) showed sensitivity to complement-mediated killing when they were cultivated at 37 degrees C (serum-sensitive) but not when they were cultivated at 20 degrees C (serum-resistant). These strains produced smooth lipopolysaccharide when they were grown at 20 degrees C and rough lipolysaccharide when cultivated at 37 degrees C. The reason for the resistance to complement-mediated killing could be that C3b is rapidly degraded (possibly because it is bound far from the cell membrane), consequently the lytic complex (C5b-9) is not formed.

We have demonstrated by using an in vitro approach that interruption of the OmpK36 porin gene by insertion sequences (ISs) is a common type of mutation that causes loss of porin expression and increased resistance to cefoxitin in Klebsiella pneumoniae. This mechanism also operates in vivo: of 13 porin-deficient cefoxitin-resistant clinical isolates of K. pneumoniae, 4 presented ISs in their ompK36 gene.

OmpK35 from Klebsiella pneumoniae is the homologue of Escherichia coli OmpF porin. Expression of OmpK35 in K. pneumoniae strain CSUB10R (lacking both OmpK35 and OmpK36) decreased the MICs of cephalosporins and meropenem ≥128-fold and decreased the MICs of imipenem, ciprofloxacin, and chloramphenicol ≥8-fold. MIC reductions by OmpK35 were 4 times (cefepime), 8 times (cefotetan, cefotaxime, and cefpirome), or 128 times (ceftazidime) higher than those caused by OmpK36, but the MICs were similar or 1 dilution lower for other evaluated agents.

Forty clonally related clinical isolates of Escherichia coli from hospitalized patients were resistant to cefoxitin (MICs, >256 μg/ml) and ceftazidime (MICs, 32 to 256 μg/ml) and were intermediate or resistant to cefotaxime (MICs, 16 to 128 μg/ml) but susceptible to both cefepime (MICs, 0.5 to 2 μg/ml) and imipenem (MICs, 0.125 to 0.25 μg/ml). Resistance to β-lactams was related to high-level production of AmpC β-lactamase and loss of OmpF porin.

Two clinical isolates of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae were noted to be less susceptible than expected to imipenem. Both were missing outer membrane proteins that serve as channels for antibiotic entry. The role of β-lactamase in resistance was investigated by eliminating the original ESBL and introducing plasmids encoding various ESBLs and AmpC β-lactamase types, by studying the effect of an increased inoculum, and by evaluating interactions with β-lactamase inhibitors. The contribution of porin deficiency was investigated by restoring a functional ompK36 gene on a plasmid. Plasmids encoding AmpC-type β-lactamases provided resistance to imipenem (up to 64 μg/ml) and meropenem (up to 16 μg/ml) in strains deficient in porins. Carbapenem resistance showed little inoculum effect, was not affected by clavulanate but was blocked by BRL 42715, and was diminished if OmpK36 porin was restored. Plasmids encoding TEM- and SHV-type ESBLs conferred resistance to cefepime and cefpirome, as well as to earlier oxyimino-β-lactams. This resistance was magnified with an increased inoculum, was blocked by clavulanate, and was also lowered by OmpK36 porin restoration. In addition, SHV-2 β-lactamase had a small effect on carbapenem resistance (imipenem MIC, 4 μg/ml, increasing to 16 μg/ml with a higher inoculum) when porins were absent. In K. pneumoniae porin loss can thus augment resistance provided either by TEM- or SHV-type ESBLs or by plasmid-mediated AmpC enzymes to include the latest oxyimino-β-lactams and carbapenems.

Klebsiella pneumoniae porin genes were analyzed to detect mutations accounting for the porin deficiency observed in many β-lactam-resistant strains. PCR and Southern blot analysis revealed the existence of a third porin gene in addition to the OmpK36 and OmpK35 porin genes previously described. This new porin gene was designated ompK37 and is present in all of the clinical isolates tested. The OmpK37 porin gene was cloned, sequenced, and overexpressed in Escherichia coli . In contrast to that of the major porins, OmpK37 porin expression was only detectable by Western blot analysis in porin-deficient β-lactam-resistant strains, suggesting strong down regulation under standard laboratory conditions. Functional characterization suggested a narrower pore for the OmpK37 porin than for K. pneumoniae porins OmpK36 and OmpK35. This correlated with the susceptibility to certain β-lactam antibiotics, since a K. pneumoniae strain expressing porin OmpK37, but not porin OmpK36 or OmpK35, was less susceptible to β-lactam antibiotics than the same strain expressing either porin OmpK36 or OmpK35.

Background: Porins are channel-forming membrane proteins that confer solute permeability to the outer membrane of Gram-negative bacteria. In Escherichia coli, major nonspecific porins are matrix porin (OmpF) and osmoporin (OmpC), which show high sequence homology. In response to high osmolarity of the medium, OmpC is expressed at the expense of OmpF porin. Here, we study osmoporin of the pathogenic Klebsiella pneumoniae (OmpK36), which shares 87% sequence identity with E. coliOmpC in an attempt to establish why osmoporin is best suited to function at high osmotic pressure.Results: The crystal structure of OmpK36 has been determined to a resolution of 3.2 Å by molecular replacement with the model of OmpF. The structure of OmpK36 closely resembles that of the search model. The homotrimeric structure is composed of three hollow 16-stranded antiparallel β barrels, each delimiting a separate pore. Most insertions and deletions with respect to OmpF are found in the loops that protrude towards the cell exterior. A characteristic ten-residue insertion in loop 4 contributes to the subunit interface. At the pore constriction, the replacement of an alanine by a tyrosine residue does not alter the pore profile of OmpK36 in comparison with OmpF because of the different course of the mainchain. Functionally, as characterized in lipid bilayers and liposomes, OmpK36 resembles OmpC with decreased conductance and increased cation selectivity in comparison with OmpF.Conclusions: The osmoporin structure suggests that not an altered pore size but an increase in charge density is the basis for the distinct physico-chemical properties of this porin that are relevant for its preferential expression at high osmotic strength., The work was supported by grants from the Swiss National Science Foundation to JPR and TS, and from Ministerio de Educación y Cultura to VJB, SHA and SA were supported by a pre-doctoral fellowship and a post-doctoral contract, respectively, from Ministerio de Educación y Cultura.

We have developed a series of vectors for easy construction of translational fusions with the phoA gene (encoding the periplasmic alkaline phosphatase, PhoA) in the three reading frames. One plasmid series carries a multiple cloning site (MCS) followed by a promoterless and leaderless 5'-truncated phoA ('phoA), which in turn is followed by a kanamycin-resistance-encoding gene (kan). Another plasmid series contains two identical inverted MCS flanking the phoA-kan cluster. These latter vectors are devised as phoA-kan cassette delivery vectors. In-frame cloning results in the production of hybrid PhoA proteins which display PhoA activity if successfully exported beyond the cytoplasmic membrane. In order to test these vectors, we have constructed hybrid PhoE::PhoA proteins, which were used to analyze the activity of the phoE promoter and identify the hybrid gene products.

We describe a method for the typing and quantitation of Klebsiella pneumoniae serotype O1 lipopolysaccharide (LPS) based on inhibition in an enzyme-linked immunosorbent assay of a reaction of known O1 LPS antigen and anti-O1 antibody by unknown LPS extracts. Serotype O1 was found in 32% of the 124 K. pneumoniae clinical isolates tested, showing that this serotype is frequent among the eight O serotypes which have been described previously.

The intracellular accumulation of norfloxacin and pefloxacin in Klebsiella pneumoniae was evaluated. The roles of lipopolysaccharide, capsule, and outer membrane proteins were not important for the intrabacterial accumulation of fluoroquinolones in isogenic strains with known outer membrane alterations. In fluoroquinolone-resistant clinical isolates also expressing GyrA alterations, an active efflux leading to decreased accumulation of the drugs enhanced their resistance to these agents.

Two porins, OmpK36 and OmpK35, have been described previously in Klebsiella pneumoniae, and they are homologous to the Escherichia coli porins OmpC and OmpF, respectively, at both the DNA and amino acid levels. Optimal resolution of the two K. pneumoniae porins by electrophoresis on polyacrylamide gels is not achieved using gel systems already described for E. coli and requires modifications of the bisacrylamide content of the resolving gels. Once resolved, identification of porins OmpK36 and OmpK35 cannot be based solely on their apparent molecular masses since in some strains the OmpK36 porin migrates faster than the OmpK35 porin, whilst in other strains OmpK35 is the faster-migrating porin. Expression of OmpK35 porin is increased in low-osmolarity medium and, combined with Western blot analysis, this allows for the identification of both porins. Application of this identification system showed that most isolates lacking expression of extended-spectrum /?-lactamases express the two porins, whereas most isolates producing these /?-lactamases express only porin OmpK36, and the OmpK35 porin is either very low or not expressed, This wo rk was supported by grants from Comision Inter- ministerial de Ciencia y Tecnologi'a (CICYT). S.H. -A., D.A., and S.A. were supported by fellowships from CICYT, a n d A.D.-S. was supported by a predoctoral fellowship from CSIC-CAROB SA.

The interaction between C1q, a subcomponent of the complement classical pathway component C1, and OmpK36, a porin protein from Klebsiella pneumoniae, was studied in a solid-phase direct-binding assay, inhibition assays with the purified globular and collagen-like regions of C1q, and cross-linking experiments. We have shown that the binding of C1q to the OmpK36 porin of the serum-sensitive strain K. pneumoniae KT707 occurs in an in vivo situation and that this binding leads to activation of the complement classical pathway and the subsequent deposition of complement components C3b and C5b-9 on the OmpK36 porin. Scatchard analysis of the binding of [125I]C1q to the OmpK36 porin showed two binding sites with dissociation constants of 1.5 and 75 nM. The decrease of [125I]C1q binding to the OmpK36 porin in buffer with increasing salt concentrations and the pIs of the C1q subcomponent (10.3) and OmpK36 porin (4.5) suggest that charged amino acids are involved in the binding phenomenon. In inhibition assays, only the globular regions of C1q inhibited the interaction between C1q and OmpK36 porin, demonstrating that C1q binds to porin through its globular region and not through the collagen-like stalks.

We have previously described an inhibition enzyme-linked immunosorbent assay method for the O typing of O1 lipopolysaccharide from Klebsiella pneumoniae which overcomes the technical problems and limitations of the classical O-typing method. In this study, we have extended the method to all of the currently recognized O types. The method was validated by studying the prototype strains that have defined the O groups by the classical tube agglutinatination O-typing method. Based on these results, we confirmed the O types of 60 of 64 typeable strains, and we propose a revised O-antigenic scheme, with minor but necessary changes, consisting of serogroups or serotypes O1, O2, O2ac, O3, O4, O5, O7, O8, and O12. Application of this typing method to 638 K. pneumoniaeclinical isolates from Denmark, Spain, and the United States from different sources (blood, urine, and others) showed that up to 80% of these isolates belong to serotypes or serogroups O1, O2, O3, and O5, independently of the source of isolation, and that a major group of nontypeable isolates, representing about 17% of the total, consists of half O+ and half O− strains. Differences were observed, however, in the prevalence of the lipopolysaccharide O types or groups, depending on the country and isolation source., This work was supported by grants from the Comisión Interministerial de Ciencia y Tecnologı́a (CICYT) of the Spanish Government. S.H.A., S.A., and D.A. were supported by predoctoral fellowships/postdoctoral contracts from CICYT.