MrkF is a component of type 3 fimbriae in Klebsiella pneumoniae

Klebsiella pneumoniae type 3 fimbriae are encoded by mrkABCDF genes which produce the major pilin subunit MrkA, chaperone MrkB, outer membrane usher MrkC, adhesin MrkD and MrkF of unknown function, respectively. RT-PCR analysis demonstrated that the mrkF gene is contained within the mrk operon. Deletion of mrkF in K. pneumoniae CG43 was found to reduce biofilm formation. A higher level of biofilm formation activity was also observed in recombinant Escherichia coli JM109[pmrkABCDF] compared to that observed for JM109[pmrkABCD]. Immunoelectron microscopy analysis of recombinant type 3 fimbriae using anti-MrkA and anti-MrkF antibody-labeled gold particles revealed that MrkF intermittently inserted into the MrkA filament. An interaction between recombinant MrkA and MrkF was demonstrated by co-immunoprecipitation analysis, further supporting the notion that MrkF is a structural component of the fimbriae. Intriguingly, the incorporation of MrkF appeared to decrease fimbrial length but increased activity of autoaggregation and biofilm formation in the bacteria JM109[pmrkABCDF]. This suggested that MrkF may play a role in assembly of the filament.     

Isolation, chemical composition, and molecular size of extracellular type II and type Ia polysaccharides of group B streptococci

Polysaccharides carrying the type II- and type Ia-specific determinants of Lancefield group B streptococci were isolated and purified by anion-exchange chromatography and gel filtration from the supernatant culture medium after growth of strain 18RS21/67/1 (type II) and strain DS/1204/78 (type Ia), respectively. The average molecular weights of these polysaccharides were 97,000 (type II) and 94,000 (type Ia), as determined by reducing end group analyses. These molecular weights were in reasonably good agreement with molecular weights determined by gel filtration at high ionic strength on calibrated columns. The polysaccharides did not cross-react with antisera specific for the other type-specific determinants or with group B-specific antisera. Their content of galactose, glucose, glucosamine, and neuraminic acid (the last two calculated as N-acetyl derivatives) accounted for over 96% of their dry weight. The two polysaccharides differed from each other (and from type III polysaccharide) in their relative content of these monosaccharides. The molar ratios of galactose, glucose, and neuraminic acid to glucosamine were 3.3:2.3:1.35:1.0 for the type II polysaccharide and 2.0:0.8:1.4:1.0 for the type Ia polysaccharides. The results obtained indicate that these extracellular type II and Ia polysaccharides contain larger amounts of neuraminic acid than can be accounted for by previously proposed structures of their repeating units.     

Evolution of Klebsiella pneumoniae with mucoid and non-mucoid type colonies within a single patient

We obtained nine Klebsiella pneumoniae isolates successively isolated from a single patient. Four pairs (M1–M4 and NM1–NM4) obtained simultaneously from the same site showed different colony types, mucoid and non-mucoid, while the final isolate (M5) was isolated alone from the blood and showed a mucoid phenotype. The whole genome of isolate M5 was sequenced de novo using the PacBio RSII system, while the others were sequenced with an Illumina Hiseq4000 and mapped to the genome sequences of M5. To identify insertions or deletions in the cps locus, we amplified and sequenced cps locus genes. We identified insertion sequence (IS) elements in several genes of the cps locus or one amino acid substitution in WcaJ in all non-mucoid isolates. Five additional amino acid alterations in RpsJ, LolE, Lon-2, PpsE, and a hypothetical protein were detected in some mucoid and non-mucoid isolates. Based on the genome data and cps locus sequences, the mucoid phenotype may have been lost or converted into the non-mucoid phenotype because of the insertion of IS elements or amino acid alterations at this locus. We inferred a within-host evolutionary scenario, in which non-mucoid variants emerged repeatedly from mucoid isolates, but may be short-lived because of their low fitness.     

New, simple medium for selective recovery of Klebsiella pneumoniae and Klebsiella oxytoca from human feces

A culture medium was developed which selectively favored the growth of Klebsiella pneumoniae and Klebsiella oxytoca in Escherichia coli-rich fecal cultures, without the use of antibiotics. The discriminative capacity of this medium was based on the presence of only two carbon sources, citrate and inositol, which can be utilized by nearly all K. pneumoniae and K. oxytoca strains but not by E. coli. The medium consisted of Simmons citrate agar (SCA) with 1% inositol (SCAI). Klebsiella strains from fecal samples subcultured on SCAI grew unhampered as yellow, dome-shaped, often mucoid colonies, whereas E. coli appeared as tiny, watery colonies. Apart from some Enterobacter strains, no other types of bacteria were found to mimic the typical appearance of klebsiellae. Recovery experiments from stool samples revealed a limiting ratio of Klebsiella to E. coli of 1:10(6) or more when samples were plated on SCAI versus ratios of 1:10(2) to 1:10(3) on blood agar or Macconkey agar. Compared with an existing Klebsiella culture method, the combination of SCA and MacConkey-inositol-carbenicillin (MIC) agar, Klebsiella yields with SCAI were not lower than those with the combination of MIC and SCA. Furthermore, the efficiency of the SCAI method was twice that of the latter combination. The SCAI plate could be a valuable tool in studies on the epidemiology of K. pneumoniae and K. oxytoca, for example in nosocomial infections, especially those concerning immunocompromised patients.     

A porin from Klebsiella pneumoniae: sequence homology, three-dimensional model, and complement binding.

A recombinant plasmid containing ompK36, the gene coding for the Klebsiella pneumoniae outer membrane protein OmpK36, was constructed by transposon mutagenesis and subcloning. Clones were identified in a cosmid library in Escherichia coli on the basis of their reaction with antiserum against the OmpK36 protein and by the presence in gel electrophoretic analysis of a band in E. coli outer membranes migrating with a mobility corresponding to 36 kDa. The ompK36-encoded protein exhibited characteristic properties of porins, such as heat modifiability and resistance to trypsin. The sequence of the gene revealed that OmpK36 is a close relative of the enterobacterial porin family, with a high degree of homology with E. coli OmpC, PhoE, and OmpF. On the basis of the structures of OmpF and PhoE porins, determined previously by X-ray analysis, it appears likely that the three-dimensional structure of OmpK36 also contains the motif of a 16-stranded beta-barrel, with long loops on one end and short turns on the other. Like the OmpC porin from E. coli, OmpK36 contains a long insertion in loop 4. The results of a binding study of complement component C1q to OmpK36 and the analysis of the OmpK36 model suggest that C1q binding sites are covered by the lipopolysaccharide core in the native porin.     

Characterization of genes encoding type 1 fimbriae of Klebsiella pneumoniae, Salmonella typhimurium, and Serratia marcescens.

With a minicell system, the organization of genes encoding type 1 fimbriae of Salmonella typhimurium, Klebsiella pneumoniae, and Serratia marcescens was determined. In all cases multiple gene products were necessary for the phenotypic expression of fimbriae; thus fimbrial expression in these strains is similar to that in Escherichia coli. The type 1 fimbrial subunit gene was detected by the ability of its product to react with specific antiserum. At least six genes were found to be involved in the expression of type 1 fimbriae by S. typhimurium, and at least four genes constituted the fimbrial gene cluster of K. pneumoniae. In the case of S. marcescens, a minimum of three detectable polypeptides was required for the production of fimbriae. Also, a gene probe consisting in part of nucleotide sequences from the E. coli fimbrial subunit gene hybridized to a discrete DNA fragment derived from the plasmid encoding K. pneumoniae fimbriae. Such a fragment was assumed to contain a gene encoding the structural component of the type 1 fimbriae. Each of the three cloned systems encoded a number of polypeptides which varied in size; thus, the organization and molecular weight of fimbrial accessory proteins of each genus were not identical.     

Construction and expression of recombinant plasmids encoding type 1 fimbriae of a urinary Klebsiella pneumoniae isolate.

The type 1 fimbriae of Klebsiella pneumoniae have been implicated as important virulence factors in mediating Klebsiella urinary infections. The chromosomally encoded fimbrial genes were cloned by a cosmid cloning technique. Further subcloning was performed with the cloning vehicles pBR322 and pACYC184, and a recombinant plasmid containing the fimbrial genes was constructed. After transformation by this plasmid, both Escherichia coli and Salmonella typhimurium were shown to express fimbriae which reacted with Klebsiella fimbrial antiserum. The approximate location of the relevant genes on the chimeric plasmid was determined by insertion of the transposable element Tn5. Hemagglutination-negative phenotypes were used to estimate the minimum size of the DNA fragment necessary to encode fimbrial biosynthesis and expression. The size of the coding region of this fragment was found to be 5.5 kilobase pairs.     

Clonal dissemination of multilocus sequence type 11 Klebsiella pneumoniae carbapenemase – producing K. pneumoniae in a Chinese teaching hospital

Klebsiella pneumoniae carbapenemase (KPC)‐producing K. pneumoniae has disseminated rapidly in China. We aimed to analyze the molecular epidemiology of four KPC‐producing K. pneumoniae strains isolated from a suspected clonal outbreak during a 3‐month period and to track the dissemination of KPC‐producing K. pneumonia retrospectively. We created antimicrobial susceptibility profiles using an automated broth microdilution system and broth microdilution methods. We screened carbapenemase and KPC phenotypes using the modified Hodge test and meropenem–boronic acid (BA) disk test, respectively. We identified β‐lactamase genes with PCR and sequencing. We investigated clonal relatedness for epidemiological comparison using pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All isolates expressed multidrug resistance and yielded positive results for the modified Hodge and meropenem‐BA disk tests. The isolates all carried bla_KPC‐2, and coproduced CTX‐M–type extended‐spectrum β‐lactamase. PFGE and MLST showed that the isolates were clonally related. The PFGE patterns of these isolates had ≥90% similarity. We found a single clone, sequence type (ST) 11, and its typical dissemination mode resembled clonal spread. The dissemination of KPC‐producing K. pneumoniae is clonally related and there is probable local transmission of a successful ST11 clone.     

First characterization in Tunisia of a TEM-15, extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolate

Klebsiella pneumoniae CH0905 strain exhibiting high-level cefotaxime resistance was isolated from a stool culture in the intensive care unit. The resistance gene responsible was shown to be located on a conjugative 60-kb plasmid designated pCH0905. The minimum inhibitory concentration (MIC) values for cefotaxime and ceftazidime of the original isolate and the transconjugates were 256 mug/ml. Isoelectric focusing of a protein preparation from the K. pneumoniae strain showed beta-lactamases with the pI values of 7.6 and 6.3. A 1,080-bp fragment amplified with PCR was cloned into the pGEM-T Easy vector. The nucleotide sequence of the complete 1,080 bp was determined. Sequence analysis revealed that the bla(TEM) gene of pCH0905 differed from bla(TEM-1) by two mutations, leading to the following amino acid substitutions: the glutamic acid residue at position 104 by lysine and the glycine residue at position 238 by serine (Ambler numbering). The association of these two mutations was described previously in TEM-15 beta-lactamase, but this is the first detection of this enzyme in Tunisia.     

Binding of a membrane proteoglycan from Klebsiella pneumoniae and its derivatives to human leukocytes.

The binding of a membrane proteoglycan from a non-encapsulated strain of Klebsiella pneumoniae (Kp-MPG) and four derivatives thereof, to human leukocytes, was investigated by indirect immunofluorescence using biotinylated F(ab')2 fragments of anti-Kp-MPG antibodies and the streptavidin-phycoerythrin amplification system in flow cytometry. Four Kp-MPG derivatives were studied: 1/ an acylpoly(1,3)galactoside (APG), 2/ an APG preparation submitted to acid hydrolysis which removed all fatty acids, but left intact the galactose chain of APG (GC-APG), 3/ a preparation obtained by mild alkaline hydrolysis, containing additional ester-linked C14 and C16 fatty acids bound to the APG molecule (EFA-APG) and 4/ a polymer of the latter compound (APG pol). Kp-MPG, APG and EFA-APG were shown to bind exclusively to monocytes at the lowest concentrations (from 0.15 to 3 microM APG). At higher concentrations, these compounds interacted with polymorphonuclear leukocytes, and with lymphocyte subsets in the following decreasing order: B cells, NK cells, CD8+ and CD4+ lymphocytes. Neither APG pol or GC-APG nor K. pneumoniae smooth LPS showed significant binding to leukocytes. However Kp-LPS treated by drastic alkaline hydrolysis displayed binding properties similar to those of APG. Removal of the ester-linked C14 and C16 fatty acids from EFA-APG did not affect the binding of the molecule. The capacity of cells from the myelomonocytic lineage to bind Kp-MPG and APG was very low in phenotypically immature cell lines (HL60 and U937) as compared with monocytes or polymorphonuclear cells. Treatment of U937 cells with interferon-gamma up-regulated their APG binding capacity along with the expression of the integrin CD 11 b and the CD 14 molecule, whereas monocytes exposed to interferon-gamma showed an increased binding of APG associated with an elevated expression of the galactose specific lectin Mac-2. The data demonstrate a preferential binding of Kp-MPG and APG to cells of the monocyte/macrophage lineage. APG binding does not involve the poly (1,3) galactose chain and the ester-linked C14 and C16 fatty acids but requires the presence of the hydrophobic part of the molecule.     

Characterization of a novel Klebsiella pneumoniae sequence type 476 carrying both bla KPC-2 and bla IMP-4

Carbapenemase-producing Klebsiella pneumoniae has recently spread rapidly throughout China. In this study, we characterized a carbapenem-resistant K. pneumoniae isolate that produced both KPC-2 and IMP-4 type carbapenemases. A clinical isolate of K. pneumoniae, resistant to both meropenem and imipenem, was recovered from a urine sample. Antibiotic susceptibility was determined using the broth microdilution method and Etest (bioMérieux, France). Pulsed-field gel electrophoresis and multilocus sequence typing (MLST) were used for gene type analysis. bla (KPC) and the encoding genes of ESBLs and plasmid-mediated AmpC enzymes were polymerase chain reaction (PCR) amplified and sequenced. Plasmids were analyzed by transformation, enzyme restriction and Southern blot. PCR analysis revealed that the isolate was simultaneously carrying bla (KPC-2), bla (IMP-4), bla (TEM-1), and bla (OKP-B) genes. MLST assigned the isolate to a novel sequence type, ST476. bla (KPC-2)-harbouring plasmids of the isolate and comparative strains had similar EcoRI and HindIII restriction maps, while IMP-4-harbouring plasmids had variable HindIII restriction maps. Coexistence of bla (KPC-2) and bla (IMP-4) was probably due to bla (IMP-4)-harbouring plasmid transmission into KPC-2-producing K. pneumoniae (ST476). The concomitant presence of these genes is alarming and poses both therapeutic and infection control problems.     

Immunogenic properties in mice of hexasaccharide from the capsular polysaccharide of Streptococcus pneumoniae type 3.

Hexasaccharide (HS) containing 3 U of cellobiuronic acid was isolated from Streptococcus pneumoniae type 3 capsular polysaccharide S3 and coupled to bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or tetanus toxoid (TT). The immunogenicity of these HS-protein conjugates in BALB/c mice was studied by measuring the production of circulating antibodies and the induction of protective immunity to viable S. pneumoniae type 3. Immunization of BALB/c mice with 0.5 micrograms of S3 resulted in the induction of immunoglobulin M (IgM) antibodies and complete protection against 25 U of a mean lethal dose of S. pneumoniae type 3 for 19 weeks after immunization. BALB/c mice immunized with 100 micrograms of HS9-BSA (containing 12 micrograms of HS) were also protected due to circulating IgM antibodies. Repeated injections with either 100 micrograms of HS9-BSA (three immunizations) or 100 micrograms of HS6-KLH (two immunizations) resulted in high levels of circulating IgG antibodies. These HS-protein conjugates induced complete protection which lasted at least 14 (HS9-BSA), 23 (HS6-KLH), or 8 (HS16-TT) weeks after the last immunization. Protection against viable S. pneumoniae type 3 could be passively transferred to nonimmunized mice by antisera containing IgM or IgG antibodies or both. Sera containing both IgM and IgG antibodies gave better protection than sera containing only IgM antibodies. The specificity of the induced protection was confirmed by challenge with the non-cross-reacting S. pneumoniae type 11.     

Klebsiella pneumoniae infection in a liver transplant patient: A case report and review of the literature

The share of Klebsiella pneumoniae in infections has been recently increasing. Multidrug-resistant strains that produce more than one antibiotic resistance mechanism are also increasingly isolated. Contamination of the organs preservation fluid occurs quite often, but the isolated microorganisms are mainly saprophytic bacteria that are part of the skin microbiota (coagulase-negative Staphylococcus, Corynebacterium spp). The following case describes a K. pneumoniae blood infection in a patient after liver transplantation. Susceptibility of the strains to chosen antimicrobials was determined using the automated method. For strain isolated from blood, it was confirmed by loop-mediated isothermal amplification of genetic material.     

Isolation and characterization of Klebsiella oxytoca strain degrading crude oil from a Tunisian off-shore oil field

A facultatively anaerobic, Gram-negative, mesophilic, moderately halotolerant, non-motile, and non-sporulated bacterium, designated strain BSC5 was isolated from an off-shore "Sercina" oil field, located near the Kerkennah island, Tunisia. Yeast extract was not required for growth. Phenotypic characteristics and phylogenetic analysis of the 16S rRNA gene sequence of strain BSC5 revealed that it was related to members of the genus Klebsiella, being most closely related to the type strain of K. oxytoca (99% sequence similarity). Strain BSC5 was capable of using aerobically the crude oil as substrate growth. The growth of strain BSC5 on crude oil was followed by measuring the OD(600 nm) and by enumeration of viable cells at different culture's time. GC-MS analysis showed that strain BSC5 was capable of degrading a wide range of aliphatic hydrocarbons from C(13) to C(30) . The biodegradation rate for n -alkanes reached 44% and 75%, after 20 and 45 days of incubation, respectively. Addition of the synthetic surfactant, Tween 80, accelerated the crude oil degradation. The biodegradation rate for n -alkanes reached 61% and 98%, after 20 and 45 days of incubation, respectively. Moreover, three aromatic compounds, p -hydroxybenzoate, protocatechuate and gentisate, were metabolized completely by strain BSC5 after 24 h, under aerobic conditions.     

Transfer of KPC-2 Carbapenemase from Klebsiella pneumoniae to Escherichia coli in a patient: first case in Europe

The first case in Europe of Klebsiella pneumoniae carbapenemase (KPC) 2 transfer from K. pneumoniae to Escherichia coli in the same patient is described. KPC-positive plasmids from the two species were identical, indicating horizontal plasmid transfer. Selection of the KPC-producing E. coli strain was triggered by therapy with meropenem.