Problems of prevalance and resistance of nosocomial Klebsiella pneumonia in hospitals of Russia]

Prevalence of Klebsiella pneumoniae among gramnegative pathogens of nosocomial infections in intensive care units of 33 hospitals of 22 towns in Russia was investigated. Antibiotic susceptibility and extended-spectrum beta-lactamase production were tested in 420 nosocomial K. pneumoniae isolates. Carbapenems (imipenem, meropenem and ertapenem) showed the highest activity. Extended-spectrum beta-lactamase production based on the phenotyping methods was revealed in 342 (81.4%) isolates. The maximum activity against the K. pneumoniae isolates producing extended-spectrum beta-lactamase was observed in imipenem and meropenem (no unsusceptible strains were isolated). 3.2% of the isolates was not susceptible to ertapenem. Differences in the activity of cefoperazone/sulbactam, amikacin, ciprofloxacin and levofloxacin against the extended-spectrum beta-lactamase producing isolates in various hospitals were recorded.     

Metabolic activities in Cyanophora paradoxa and its cyanelles

Isolated cyanelles of Cyanophora paradoxa perform photosystem I and II dependent Hill reactions. The photosynthetic electron transport of the cyanelles does not show special features uncommon in cyanobacteria or chloroplasts of red algae. A preparation of cyanelles performs photosynthetic O₂-evolution with approximately 1/3 of the rate of intact Cyanophora, in only, however, the first three minutes of the experiment. All attempts to stabilize the CO₂-fixation activity of isolated cyanelles failed. Isolated cyanelles do not perform KCN-sensitive O₂-uptake, indicating that respiratory cytochrome oxidase is lacking in cyanelles. O₂-consumption by crude extracts from Cyanophora is inhibited by KCN when N-tetramethyl-p-phenylenediamine/ascorbate or NADH but not NADPH are supplied as the electron donors in contrast to the situation in cyanobacteria. These findings suggest that cyanelles do not respire. It is concluded that cyanelles are not so much related to cyanobacteria as formerly believed, but share many properties with chloroplasts of eukaryotic cells.Abbreviations Chl chlorophyll - DCPIP dichlorophenol-indophenol - TMPD N-tetramethyl-p-phenylenediamine To whom correspondence should be addressed     

Isolation of the plastid FtsZ gene from Cyanophora paradoxa (Glaucocystophyceae, Glaucocystophyta)

Plastids of glaucocystophytes are termed cyanelles and retain primitive features, such as a peptidoglycan wall. We isolated a full‐length prokaryotic plastid division gene, FtsZ, from the glaucocystophyte alga Cyanophora paradoxa Korshikov (CpFtsZ‐cy). CpftsZ‐cy has a chloroplast‐targeting signal at the N‐teminus. Immunofluorescence microscopy showed that CpFtsZ‐cy forms a ring‐like structure at the division plane of cyanelles.     

Incidence of two virulence factors (aerobactin and mucoid phenotype) among 190 clinical isolates of Klebsiella pneumoniae producing extended-spectrum β-lactamase

Because outbreaks of multiple-resistant Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases were recently observed in French hospitals, the presence of virulence factors was examined for (i) phenotype by bioassay for aerobactin production and by culture for the mucoid phenotype, and (ii) genotype using intragenic probes of respectively 2-kb BglII and 235-bp BamHI-BglII fragments and dot-blotting among 190 unreplicated K. pneumoniae clinical isolates issued from 25 French hospitals and producing different types of extended-spectrum beta-lactamases (TEM-related enzymes: TEM-3, TEM-4, CAZ-1, CAZ-2, TEM-8, or SHV-related enzymes: SHV-2, SHV-3, SHV-4). Only 3.7% and 7% of K. pneumoniae isolates produced aerobactin and mucoid phenotypes respectively, unrelated to type of beta-lactamase. Only 2% had both factors. No discordance was reported according to the detection method tested. The low prevalence of such virulence factors seems to indicate they were not involved in dissemination of nosocomial K. pneumoniae isolates producing an extended-spectrum beta-lactamase.     

Protein and isozyme patterns of the cyanelles of Glaucocystis nostochinearum compared with Anacystis nidulans

The cyanelles of Glaucocystis nostochinearum were isolated after disruption of the algal cells by sonication. The aqueous extracts from these cyanelles were subjected to molecular filtration and electrophoresis on polyacrylamide gels. By comparison with extracts of a unicellular Chroococcalian alga, Anacystis nidulans treated in the same way only about half the number of protein bands were found. The proteins were water-soluble with a MW in excess of 10 000. Three protein-pigment complexes were detected in Anacystis. Two of these (phycoerythrin and phycocyanin) were not present in the cyanelles of Glaucocystis. Three branching glucosyltransferase isozymes capable of converting amylopectin to phytoglycogen were present in the Cyanophyte; only two branching isozymes with typical Chlorophycean ‘Q’ activity were present in the cyanelles of Glaucocystis. It seems improbable that the cyanelles of this alga are endosymbiotic blue-green algae; rather, they may represent some intermediate stage in the development of the chloroplast of green algae.     

Expression of beta-lactamase by recombinant Escherichia coli strains containing plasmids of different sizes--effects of pH, phosphate, and dissolved oxygen

The characteristics of growth and synthesis of plasmid-encoded protein were studied for strains of recombinant E. coli JM103 which carried the beta-lactamase gene on plasmids of different sizes. The plasmids used included the vector pUC8 and its recombinant derivatives containing varying-sized inserts of Drosophila DNA (not expressed in E. coli). Luria broth (LB) and a minimal medium (M9) supplemented in some cases with additional inorganic phosphate were used as growth media. There was no evidence of segregational instability in these experiments, where no antibiotic selection pressure was employed. Responses of the recombinant strains to variations in environmental parameters including pH, phosphate concentration in the medium, and aeration rate were examined. While the cell growth rate in LB decreased with pH in the range 7.0-8.0, the bulk beta-lactamase activity was maximized at an intermediate pH. The recombinant cell growth rate decreases with increasing plasmid size in the minimal medium, while such decrease is not significant when a rich medium such as LB is used. There is an intermediate plasmid size in the range studied (2.7-8.7 kb), at which beta-lactamase activity is maximum. While reduction in aeration rate (which determines the dissolved oxygen level) is detrimental for cell growth, it is beneficial for beta-lactamase synthesis. The bulk beta-lactamase activity therefore exhibits a maximum with respect to aeration rate. Cell growth and beta-lactamase production are affected in a similar manner by phosphate concentration in the minimal medium and therefore both are maximized at the same phosphate concentration. This investigation demonstrates clearly how the production of a recombinant plasmid-encoded protein can be maximized by proper manipulation of culture conditions and how it is affected by plasmid size.     

Transketolase from Cyanophora paradoxa: In Vitro Import into Cyanelles and Pea Chloroplasts and a Complex History of a Gene Often, But Not Always, Transferred in the Context of Secondary Endosymbiosis

ABSTRACT. The glaucocystophyte Cyanophora paradoxa is an obligatorily photoautotrophic biflagellated protist containing cyanelles, peculiar plastids surrounded by a peptidoglycan layer between their inner and outer envelope membranes. Although the 136-kb cyanelle genome surpasses higher plant chloroplast genomes in coding capacity by about 50 protein genes, these primitive plastids still have to import >2,000 polypeptides across their unique organelle wall. One such protein is transketolase, an essential enzyme of the Calvin cycle. We report the sequence of the pre-transketolase cDNA from C. paradoxa and in vitro import experiments of precursor polypeptides into cyanelles and into pea chloroplasts. The transit sequence clearly indicates the localization of the gene product to cyanelles and is more similar to the transit sequences of the plant homologues than to transit sequences of other cyanelle precursor polypeptides with the exception of a cyanelle consensus sequence at the N-terminus. The mature sequence reveals conservation of the thiamine pyrophosphate binding site. A neighbor-net planar graph suggests that Cyanophora , higher plants, and the photosynthetic protist Euglena gracilis acquired their nuclear-encoded transketolase genes via endosymbiotic gene transfer from the cyanobacterial ancestor of plastids; in the case of Euglena probably entailing two transfers, once from the plastid in the green algal lineage and once again in the secondary endosymbiosis underlying the origin of Euglena's plastids. By contrast, transketolase genes in some eukaryotes with secondary plastids of red algal origin, such as Thalassiosira pseudonana , have retained the pre-existing transketolase gene germane to their secondary host.     

Protein translocation into and within cyanelles (Review)

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria in morphology, pigmentation and, especially, in the presence of a peptidoglycan wall situated between the inner and outer envelope membranes. However, it is now clear that cyanelles in fact are primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high plastid gene content of the Porphyra purpurea rhodoplast and the peptidoglycan wall of glaucocystophyte cyanelles. This means that the import apparatus of all primary plastids should be homologous. Indeed, heterologous in vitro import can now be shown in both directions, provided a phenylalanine residue essential for cyanelle import is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved explaining the efficient heterologous import of native precursors from C. paradoxa. With respect to conservative sorting in cyanelles, both the Sec and Tat pathways could be demonstrated. Another cyanobacterial feature, the dual location of the Sec translocase in thylakoid and inner envelope membranes, is also unique to cyanelles. For the first time, protease protection of internalized lumenal proteins could be shown for cyanobacteria-like, phycobilisome-bearing thylakoid membranes after import into isolated cyanelles.     

Close evolutionary relationship between the chromosomally encoded beta-lactamase gene of Klebsiella pneumoniae and the TEM beta-lactamase gene mediated by R plasmids

Sixty-three percent homology of nucleotide sequence and 67% homology of deduced amino acid sequence were found between the chromosomally encoded beta-lactamase gene of Klebsiella pneumoniae and the TEM beta-lactamase of transposon Tn3. Moreover, 22 out of 24 amino acid residues are identical around the predicted active site. It is therefore suggested that these two kinds of beta-lactamases share a common evolutionary origin. The 0.5 kb DNA fragment of the cloned gene hybridized specifically with the chromosomal DNA of all the K. pneumoniae strains tested which had been isolated in Japan, USA and Europe.     

Membrane permeability modifications are involved in antibiotic resistance in Klebsiella pneumoniae

Two Klebsiella pneumoniae strains selected according to their high cross-resistance pattern to cephalosporins were characterized by (i) outer membrane protein content such as OmpA or nonspecific porins, (ii) MICs of various cephalosporins and unrelated antibiotics, (iii) beta-lactamase production, and (iv) active efflux of fluoroquinolones. An association of porin deficiency and beta-lactamase production induced a noticeable cephalosporin resistance. In addition to these mechanisms, the presence of an active efflux participating in high-level fluoroquinolone resistance was identified in one strain. The decrease of antibiotic uptake associated with efflux explains the Klebsiella adaptation against the drugs present in the environment.     

Protein translocation into and within cyanelles (review)

The cyanelles of the glaucocystophyte alga Cyanophora paradoxa resemble endosymbiotic cyanobacteria in morphology, pigmentation and, especially, in the presence of a peptidoglycan wall situated between the inner and outer envelope membranes. However, it is now clear that cyanelles in fact are primitive plastids. Phylogenetic analyses of plastid, nuclear and mitochondrial genes support a single primary endosymbiotic event. In this scenario cyanelles and all other plastid types are derived from an ancestral photosynthetic organelle combining the high plastid gene content of the Porphyra purpurea rhodoplast and the peptidoglycan wall of glaucocystophyte cyanelles. This means that the import apparatus of all primary plastids should be homologous. Indeed, heterologous in vitro import can now be shown in both directions, provided a phenylalanine residue essential for cyanelle import is engineered into the N-terminal part of chloroplast transit peptides. The cyanelle and likely also the rhodoplast import apparatus can be envisaged as prototypes with a single receptor showing this requirement for N-terminal phenylalanine. In chloroplasts, multiple receptors with overlapping and less stringent specificities have evolved explaining the efficient heterologous import of native precursors from C. paradoxa. With respect to conservative sorting in cyanelles, both the Sec and Tat pathways could be demonstrated. Another cyanobacterial feature, the dual location of the Sec translocase in thylakoid and inner envelope membranes, is also unique to cyanelles. For the first time, protease protection of internalized lumenal proteins could be shown for cyanobacteria-like, phycobilisome-bearing thylakoid membranes after import into isolated cyanelles.     

Chromosome-encoded beta-lactamases of Citrobacter diversus. Interaction with beta-iodopenicillanate and labelling of the active site.

Both forms of the chromosome-encoded beta-lactamase of Citrobacter diversus react with beta-iodopenicillanate at a rate characteristic of class A beta-lactamases. The active site of form I was labelled with the same reagent. The sequence of the peptide obtained after trypsin hydrolysis is identical with that of a peptide obtained in a similar manner from the chromosome-encoded beta-lactamase of Klebsiella pneumoniae.     

Construction of multicopy expression vectors for regulated over-production of proteins in Klebsiella pneumoniae and other enteric bacteria

A number of expression vectors have been constructed to allow over-production of selected gene products in Klebsiella pneumoniae and other enteric bacteria. The plasmids use the strong hybrid trp-lac (tac) promoter for gene expression, which is regulated by the lacIQ allele of the lac repressor carried on the vector. This provides very tight regulation of gene expression, which is important for over-production of proteins which may be detrimental to cell growth. The vectors carry the standard mp18 cloning nest in which all the restriction sites are unique to the plasmid (with the exception of EcoRI in pDK7). Derivatives were constructed carrying kanamycin, chloramphenicol or ampicillin resistance as selectable markers, the first two of which are advantageous in K. pneumoniae due to the high inherent beta-lactamase activity of this organism.     

Bactericidal activities of rat defensins and synthetic rabbit defensins on Staphylococci, Klebsiella pneumoniae (Chedid, 277, and 8N3), Pseudomonas aeruginosa (mucoid and nonmucoid strains), Salmonella typhimurium (Ra, Rc, Rd, and Re of LPS mutants) and Escherichia coli.

Rat defensins were purified and tested for in vitro bactericidal assay against gram-positive and gram-negative bacteria. Staphylococcus aureus (209P, Cowan I, Smith diffuse and Smith compact) were resistant to defensins, whereas Staphylococcus epidermidis, Staphylococcus saprophyticus, Micrococcus lysodeikticus and Bacillus subtilis were less sensitive. Gram-negative bacteria, such as Pseudomonas aeruginosa (mucoid and K) and Klebsiella pneumoniae (Chedid, 277, and 8N3 which were heavily capsulated, moderately capsulated and noncapsulated, respectively) were all very sensitive to defensins and killed within 20 min. Escherichia coli was moderately sensitive and the rough mutants of lipopolysaccharide (LPS) of Salmonella typhimurium LT2, such as Ra, Rc, Rd, and Re were equally sensitive to defensins, being killed within 40 min. Lysozyme did not show any bactericidal activity except against M. lysodeikticus and B. subtilis, whereas it enhanced the bactericidal activity of defensins against P. aeruginosa, E. coli, and K. pneumoniae and suppressed the killing activity of defensins against S. typhimurium and S. aureus. With regard to the three synthetic rabbit defensins, NP1, NP4, and NP5, NP1 showed strong bactericidal activity against K. pneumoniae 277, comparable to that of rat defensins. Neither NP4 nor NP5 showed any bactericidal activity, while NP5 rather enhanced the bactericidal activity of NP1 against K. pneumoniae 277.     

Exchange of Metabolites in Cyanophora paradoxa and its Cyanelles*

The flagellate Cyanophora paradoxa contains blue-greenish, organelle-like inclusions termed cyanelles which perform photosynthetic CO₂-fixation in place of chloroplasts. By the use of the HPLC-technique, Cyanophora was shown to form glucose, sucrose, maltose, mannitol, ribose, glycerol and trehalose. Extracts from the whole organism and from the eucaryotic host, but not from the cyanelles, convert ¹⁴C-labelled UDP-glucose to polyglucan. Synthesis of sucrose from UDP-glucose and fructose-6-P or fructose could not be demonstrated in any extract from Cyanophora. The transfer of metabolites into cyanelles was monitored by the silicone oil filtering technique. The solute spaces for ¹⁴C-labelled sorbitol and ³H₂O were the same indicating that sorbitol freely penetrated the plasma membrane of cyanelles in contrast to the situation found in chloroplasts. The measurements of the solute spaces for the different compounds showed that maltose and sucrose were not accumulated by isolated cyanelles. Other compounds like fructose, fucose, glutamine or glycine had intermediate sizes of their solute spaces. Cyanelles apparently possess a rapidly transporting glucose carrier and not a malate/oxaloacetate shuttle and also not an ATP/ADP translocator. The carrier composition at the plasma membrane of cyanelles and at the inner envelope membrane of chloroplasts seems to be totally different.     

GLAUCOSPHAERA VACUOLATA,ITS ULTRASTRUCTURE AND PHYSIOLOGY1,2

Glaucosphaera vacuolata was recently rediscovered, and isolated by Dr. Richard Starr of Indiana University Physiological and ultrastructural studies have been conducted on this cyanome and are reported for the first time. Glaucosphaera has been found to have low rates of photosynthesis, respiration, and Hill activity. Unusual features noted include the presence of R-phycocyanin as the major accessory pigment, phycobilisomes, and eyeshot-like structures in the cyanelles.     

Penetration of β-lactamase inhibitors into the periplasm of Gram-negative bacteria

The effectiveness of a beta-lactamase inhibitor/beta-lactam combination against Gram-negative pathogens depends on many interplaying factors, one of which is the penetration of the inhibitor across the outer membrane. In this work we have measured the relative penetrations of clavulanic acid, sulbactam, tazobactam and BRL 42715 into two strains of Escherichia coli producing TEM-1 beta-lactamase, two strains of Klebsiella pneumoniae producing either TEM-1 or K-1, and two strains of Enterobacter cloacae each producing a Class C beta-lactamase. It was shown that clavulanic acid penetrated the outer membranes of all these strains more readily than the other beta-lactamase inhibitors. For the strains of E. coli and K. pneumoniae clavulanic acid penetrated approximately 6 to 19 times more effectively than tazobactam, 2 to 9 times more effectively than sulbactam and 4 to 25 times more effectively than BRL 42715. The superior penetration of clavulanic acid observed in this study is likely to contribute to the efficacy of clavulanic acid/beta-lactam combinations in combating beta-lactam resistant bacterial pathogens.     

Prevalence of extended-spectrum beta-lactamases produced by nosocomial isolates of Enterobacteriaceae in Trakya University Hospital,Turkey

The prevalence of extended-spectrum beta-lactamase (ESBL) production by 194 nosocomial isolates of Enterobacteriacea recovered from 1995 to 1999 was investigated. The ESBL production was determined by the double-disk synergy test and was confirmed by the E-test ESBL strip. Twenty-three isolates (21 Klebsiella pneumoniae, one Escherichia coli, one Providencia rettgeri) were found as ESBL-producers (11.8%). These isolates were also usually resistant to non-betalactam antibiotics. Most of them contained a beta-lactamase with a pI of 7.6. All the strains conjugally transferred their ESBLs to recipient E. coli. Contrary to others, ESBL-producing K. pneumoniae strains isolated in 1999 were resistant to ciprofloxacin, and had the identical plasmid profiles suggestive of an outbreak. Ciprofloxacin resistance in these strains could not be transferred. In conclusion, K. pneumoniae was the main ESBL-producing species among nosocomial isolates of Enterobacteriacae in our hospital.     

Separation of plasmid-mediated extended spectrum β-lactamases by fast protein liquid chromatography (FPLC system)

We have devised a reliable procedure for the separation of three beta-lactamases of isoelectric focusing points (pI), 5.4, 6.5, and 7.9 by Fast Protein Liquid Chromatography (FPLC System). All of these enzymes were transferable and originated from a ceftazidime and cefotaxime resistant Klebsiella pneumoniae isolated in Bombay, India. The complete separation of the enzymes, achievable by this method, allowed each of the different individual beta-lactamases to be characterized biochemically. This analysis revealed that the enzymes of pI 6.5 and pI 7.9 hydrolysed ceftazidime and cefotaxime, and were responsible for the resistance of K. pneumoniae, and its Escherichia coli J53-2 transconjugant to third generation cephalosporins. The enzyme of pI 5.4 was the TEM-1 beta-lactamase. The beta-lactamase of pI 7.9 appears quite different from any previously reported third generation cephalosporin hydrolysing beta-lactamase, and consequently given the preliminary designation DJP-1. This is also the first example of extended spectrum hydrolysing beta-lactamases found in Asia.     

Indicator method of determining the beta-lactamase-inhibiting activity of various compounds

An indicator for determination of beta-lactamase inhibitory activity of various compounds was developed. The method is based on the direct contact of beta-lactamase with the compounds tested. It excludes the use of test-bacteria and provides recording in the data on the day of the experiment. The indicator method enables the detection of the beta-lactamase inhibitory properties of both beta-lactamase inhibitors and beta-lactam antibiotics, not subjected to destruction by beta-lactamases. The method is likely to be fit for detection of atypical beta-lactams having beta-lactam groups in their molecules (bleomycin group). Antibiotics not belonging to the group of beta-lactams, such as gentamicin, sisomicin, lincomycin and fusidin showed no beta-lactamase inhibitory activity under the conditions of the indicator method. The use of the indicator method provided determination of the inhibitory activity with respect to penicillinase of Bac. licheniformis 749/C in 30 (8.5 per cent) out of 350 fermentation broths of actinomycetes.