Effect of bacteriophage P1 lysogeny on lipopolysaccharide composition and the lambda receptor of Escherichia coli.

The outer membrane of Escherichia coli was altered as a consequence of lysogeny by bacteriophages P1 and P1 cmts. The predominant change was a reduction in the size of lipopolysaccharide to a heptose-deficient form. P1 cmts lysogens were still sensitive to several bacteriophages but were resistant to lambda vir. Neither whole cells nor solubilized outer membranes from P1 cmts lysogens were able to inactivate lambda vir, and 32P-labeled lambda vir was unable to adsorb to P1 cmts lysogens. P1 cmts lysogens were also affected in maltose transport. The level of periplasmic maltose-binding protein was reduced somewhat, but there was no significant reduction in the level of the outer membrane lambda receptor (LamB). These membrane abnormalities were all corrected in strains cured of P1 cmts. It is suggested that P1 cmts affects lipopolysaccharide biosynthesis by a phage conversion mechanism and consequently the function of the lambda receptor.     

P1CMts lysogeny and P1 sensitivity on Klebsiella pneumoniae

Abstract The lipopolysaccharide (LPS) of Klebsiella pneumoniae was altered as a consequence of P1CM ts lysogeny by a reduction in size to a heptose-deficient form. P1CM ts lysogens were sensitive to P1 vir and K. pneumoniae phages. P1CM ts -cured strains were sensitive to P1CM ts and P1 vir and P1CM ts lysogens were likewise able to inactivate P1 vir . Concomitantly, the wild type showed a smooth LPS, while P1CM ts lysogens and P1CM ts -cured strains showed a rough LPS (heptose-deficient form).
It is suggested that P1CM ts and P1 vir , are only able to infect K. pneumoniae by selecting a LPS-deficient mutant (chemotype Re).     

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.     

Use of bacteriophage Mu to isolate deletions in the his-nif region of Klebsiella pneumoniae.

Klebsiella pneumoniae M5a1 is naturally resistant to infection by bacteriophage Mu. Mutants of K. pneumoniae sensitive to Mu infection were isolated and found to support both lytic and lysogenic development of Mu. K. pneumoniae lysogens containing a heat-inducible Mu prophage integrated in his were isolated. Strains carrying deletions extending from his into nif were obtained after heat treatment of these lysogens. Such deletions should be useful for determining the map order and cistronic organization of the nif genes.     

Conditionally replicating plasmid vectors that can integrate into the Klebsiella pneumoniae chromosome via bacteriophage P4 site-specific recombination.

P4 is a satellite phage of P2 and is dependent on phage P2 gene products for virion assembly and cell lysis. Previously, we showed that a virulent mutant of phage P4 (P4 vir1) could be used as a multicopy, autonomously replicating plasmid vector in Escherichia coli and Klebsiella pneumoniae in the absence of the P2 helper. In addition to establishing lysogeny as a self-replicating plasmid, it has been shown that P4 can also lysogenize E. coli via site-specific integration into the host chromosome. In this study, we show that P4 also integrates into the K. pneumoniae chromosome at a specific site. In contrast to that in E. coli, however, site-specific integration in K. pneumoniae does not require the int gene of P4. We utilized the alternative modes of P4 lysogenization (plasmid replication or integration) to construct cloning vectors derived from P4 vir1 that could exist in either lysogenic mode, depending on the host strain used. These vectors carry an amber mutation in the DNA primase gene alpha, which blocks DNA replication in an Su- host and allows the selection of lysogenic strains with integrated prophages. In contrast, these vectors can be propagated as plasmids in an Su+ host where replication is allowed. To demonstrate the utility of this type of vector, we show that certain nitrogen fixation (nif) genes of K. pneumoniae, which otherwise inhibit nif gene expression when present on multicopy plasmids, do not exhibit inhibitory effects when introduced as merodiploids via P4 site-specific integration.     

Antibiotic susceptibility and genotype patterns of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from urinary tract infected patients

Thirty nine isolates of Escherichia coli, twenty two isolates of Klebsiella pneumoniae and sixteen isolates of Pseudomonas aeruginosa isolated from urinary tract infected patients were analyzed by antimicrobial susceptibility typing and random amplified polymorphic DNA (RAPD)-PCR. Antibiotic susceptibility testing was carried out by microdilution and E Test methods. From the antibiotic susceptibility, ten patterns were recorded (four for E. coli, three for K. pneumoniae and three for P. aeruginosa respectively). Furthermore, genotyping showed seventeen RAPD patterns (seven for E. coli, five for K. pneumoniae and five for P. aeruginosa respectively). In this study, differentiation of strains of E. coli, K. pneumoniae and P. aeruginosa from nosocomial infection was possible with the use of RAPD.     

2009年度宁波市妇儿医院主要病原菌的临床分布及耐药性分析

目的了解宁波市妇儿医院主要病原菌的临床分布及耐药性分析。方法血液培养采用法国生物梅里埃公司的BacT/Alert3D,菌株鉴定采用法国生物梅里埃公司的VITEK 60分析仪,药敏试验采用K-B法,纸片扩散确证试验检测ESBLs。结果前10位细菌构成比依次是大肠埃希菌(13.4%)、白色念珠菌(8.7%)、阴道加德纳菌(7.8%)、表皮葡萄球菌(6.9%)、金黄色葡萄球菌(5.6%)、肺炎克雷伯菌(4.9%)、鲍曼复合醋酸钙不动杆菌(3.9%)、粪肠球菌(D群)(3.1%)、铜绿假单胞菌(2.7%)和溶血葡萄球菌(2.0%)。大肠埃希菌539株中产ESBLs阳性率为52.5%,肺炎克雷伯菌195株中产ESBLs阳性率为45.2%。大肠埃希菌主要分离于尿液,其次是脓液/切口。肺炎克雷伯菌在痰及咽拭子中所占比例最高。46株铜绿假单胞菌和11株鲍曼复合醋酸钙不动杆菌对亚胺培南耐药。结论对产ESBLs的肺炎克雷伯菌和大肠埃希菌、耐甲氧西林金黄色葡萄球菌(MRSA)和耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)、耐亚胺培南的鲍曼复合醋酸钙不动杆菌和铜绿假单胞菌,应加强隔离预防,控制在医院内的扩散,减少耐药菌株产生。     

Prevalence and antimicrobial resistance of extended-spectrum beta-lactamases-producing Escherichia coli and Klebsiella pneumoniae strains isolated in a university hospital in Split, Croatia.

The prevalence of Escherichia coli and Klebsiella pneumoniae that produce extended-spectrum b-lactamases (ESBL) was investigated in patients of a university hospital in Split, Croatia. Patients were grouped according to age (pediatric vs. adult), antibiotic type, and hospital ward. From Jan. 2001 to Dec. 2002, the susceptibility of E. coli and K. pneumoniae isolates to antimicrobials was tested. ESBL production was assayed using the double-disk synergy test. ESBL-producing E. coli and K. pneumoniae were detected in all sites of infection sampled. The percentages of ESBL-positive isolates were higher in the pediatric wards than in the adult wards. The antibiotics most commonly prescribed to patients in all hospital wards belonged to the third-generation cephalosporin group. Among ESBL producers, E. coli isolates were more resistant to aminoglycosides, but less resistant to ciprofloxacin and cotrimoxazole. Resistance of E. coli and K. pneumoniae to ciprofloxacin was exclusively found in isolates from adult patients. None of the isolates, regardless of ESBL production, was resistant to carbapenemes. In addition, the prevalence and antimicrobial resistance of ESBL-producing E. coli and K. pneumoniae isolates differed between pediatric and adult patients.     

Fine structures of the capsules of Klebsiella pneumoniae and Escherichia coli K1.

The fine structures of the capsules of Klebsiella pneumoniae and Escherichia coli were determined by the rapid-freezing technique. The capsular layer was seen as a densely packed accumulation of fine fibers. The thickness of the capsule was approximately 160 nm in K. pneumoniae and less than 10 nm in E. coli K1. Two layers were observed in the Klebsiella capsule in which the arrangements of the fibers were different. The inner layer of the capsule was formed by a palisade of thick and dense bundles of the fibers standing at right angles on the surface of the outer membrane. In the outer layer these thick bundles of fibers loosened into fine fibers which spread over the bacterial surface, forming a fine network structure.     

5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenases of Escherichia coli C and Klebsiella pneumoniae M5a1 show very high N-terminal sequence homology

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMS) dehydrogenase from Escherichia coli C and Klebsiella pneumoniae M5a1 have been purified and some of their properties studied. The apparent Km values for NAD and CHMS were 11.7 +/- 1.5 microM and 5.2 +/- 1.9 microM, respectively, for the K. pneumoniae enzyme, and 19.5 +/- 2.7 microM and 9.2 +/- 1.4 microM, respectively, for the E. coli enzyme. Both enzymes were optimally active at pH 7.5 in sodium phosphate buffer. They had subunit molecular weights of 52,000 (+/- 1000) and the native enzymes appeared to be dimers of identical subunits. The first 20 residues of their N-terminal amino acid sequences were 90% homologous. A degenerate oligonucleotide probe constructed to a six amino acid sequence common to both enzymes gave strong hybridization with DNA from E. coli strains B and W as well as with E. coli C and K. pneumoniae but little or no hybridization to DNA from E. coli K12 or Pseudomonas putida.     

Construction of intergeneric hybrids using bacteriophage P1CM: transfer of the Klebsiella aerogenes ribitol dehydrogenase gene to Escherichia coli.

Study of many of the interesting properties of Klebsiella aerogenes is limited by the lack of a well-characterized genetic system for this organism. Our investigations of the evolution of the enzyme ribitol dehydrogenase (EC 1.1.1.56) in K. aerogenes would be greatly facilitated by the availability of such a system, and we here report two approaches to developing one. We have isolated mutants sensitive to the coliphage P1, which will efficiently tranduce genetic markers between such sensitive strains and which will thus make detailed mapping studies possible. Derivatives of K. aerogenes lysogenic for P1 can be readily isolated by using the specialized transducing particle P1CMclr100. Bacteria lysogenic for this phage are chloramphenicol resistant and temperature sensitive. Phage particles produced by temperature induction of such lysogens can be used to transfer K. aerogenes genes to the natural host of P1 phage. Escherichia coli. We have used this method to prepare derivatives of E. coli K-12 carrying the K. aerogenes genes conferring the ability to metabolize the pentitols ribitol and D-arabitol. We have shown that these E. coli-K. aerogenes hybrids synthesize a ribitol dehydrogenase with the properties of the K. aerogenes enzyme and have mapped the position of the transferred gene on the E. coli chromosome. The ramifications of this methodology are discussed.     

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae and Escherichia coli

A rapid and efficient method for plasmid transformation of Klebsiella pneumoniae M5a1 and Escherichia coli K12 has been developed. The method, which uses a freeze-thaw cycle in the presence of CaCl2 to facilitate DNA uptake, is substantially more efficient for K. pneumoniae M5a1 than the conventional transformation procedure for E. coli. The simplicity and speed of the method makes it very attractive for routine transformation of K. pneumoniae M5a1 and E. coli K12.     

Bacteria recovered without subculture from infected human urines expressed iron-regulated outer membrane proteins

Abstract Twelve enteric bacterial strains were recovered by differential centrifugation of urines which were collected from clinically diagnosed and microbiologically confirmed cases of urinary tract infection. The outer membrane protein (OMP) profiles of the clinical isolates were then analysed by sodiumdodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). It was found that 5 of the 12 isolates (3 Escherichia coli strains, 1 Klebsiella pneumoniae and 1 Proteus mirabilis strain) expressed 2 or more high M _r proteins in the range of 66000 to 85000. These high M _r proteins were expressed by the same organisms during growth in vitro in iron-restricted conditions but not in iron-sufficient media.
In addition, it was found that the major outer membrane proteins expressed by the clinical isolates varied considerably and that, in many cases, fresh isolates expressed fewer porin proteins than the same bacterial strains after growth in vitro in trypticase soy broth. This is thus the first evidence the E. coli, K. pneumoniae and P. mirabilis grow under iron-restricted conditions in the urinary tract of humans and that the outer membrane protein profile of clinical isolates differ from in vitro grown bacteria.     

Expression of nitrogen fixation genes in foreign hosts. Assembly of nitrogenase Fe protein in Escherichia coli and in yeast

In Klebsiella pneumoniae, the nifH gene encodes the Fe protein (Kp2) polypeptide that is assembled into a homodimer responsible for the reduction of nitrogenase. Escherichia coli or the yeast Saccharomyces cerevisiae, transformed with the K. pneumoniae nifH gene in suitable expression vectors, synthesize the Fe protein polypeptide. This study examines the assembly of the nifH gene product into its characteristic dimeric structure in E. coli and in yeast. Immunoblotting methods, as well as 55Fe2- labeling of K. pneumoniae were employed to detect native nitrogenase components in cell lysates. E. coli and yeast transformants contained a protein similar to native Kp2 in its immunoreactivity, apparent molecular weight, and lability in the presence of oxygen or MgATP. While in E. coli the co-introduction of nifH and nifM resulted in enhanced levels of the nifH product, it appears that the nifH gene product alone is sufficient for the assembly of an Fe protein-like structure in foreign prokaryotic and eukaryotic hosts.     

The effect of inulin on the biological properties of enterobacteria

The influence of inulin on some biological properties of Enterobacteriaceae, both pathogenic (Salmonella) and opportunistic (Klebsiella, Escherichia coli) was experimentally determined in vitro. The study revealed that inulin stimulated the production of colicin and suppressed the hemolytic activity in E. coli. The effect produced by inulin on the antilysozyme activity (ALA) of Enterobacteriaceae was different: in S. typhimurium ALA was decreased and in most K. pneumoniae strains the expression of this sign was enhanced.     

Inactivation of bacteriophages by protein E, a new major membrane protein isolated from an Escherichia coli mutant.

Pure protein E, obtained after diethylaminoethyl-cellulose chromatography of ethylenediaminetetraacetic acid-Triton X-100-solubilized outer membrane proteins of Escherichia coli strain JF694, inactivated bacteriophage K3. Lipopolysaccharide enhanced bacteriophage inactivation. Antibody prepared against purified protein E protected bacteriophage K3 from inactivation by protein E. Bacteriophage K3 used a major outer membrane protein, protein II*, as part of its receptor. We conclude that proteins E and II* have a common region which interacts with bacteriophage K3. Protein E also inactivated two recently described bacteriophages, TC45 and TC23, that use protein E as at least part of their receptor.     

Specificities of reactivating factors for adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase

Adenosylcobalamin-dependent glycerol and diol dehydratases undergo inactivation by the physiological substrate glycerol during catalysis. In the permeabilized cells of Klebsiella pneumoniae, Klebsiella oxytoca, and recombinant Escherichia coli, glycerol-inactivated glycerol dehydratase and diol dehydratase are reactivated by their respective reactivating factors in the presence of ATP, Mg2+, and adenosylcobalamin. Both of the reactivating factors consist of two subunits. To examine the specificities of the reactivating factors, their genes or their hybrid genes were co-expressed with dehydratase genes in E. coli cells in various combinations. The reactivating factor of K. oxytoca for diol dehydratase efficiently cross-reactivated the inactivated glycerol dehydratase, whereas the reactivating factor of K. pneumoniae for glycerol dehydratase hardly cross-reactivated the inactivated diol dehydratase. Both of the two hybrid reactivating factors rapidly reactivated the inactivated glycerol dehydratase. In contrast, the hybrid reactivating factor containing the large subunit of the glycerol dehydratase reactivating factor hardly reactivated the inactivated diol dehydratase. These results indicate that the glycerol dehydratase reactivating factor is much more specific for the dehydratase partner than the diol dehydratase reactivating factor and that a large subunit of the reactivating factors principally determines the specificity for a dehydratase.     

Synthesis of a Klebsiella pneumoniae O-antigen heteropolysaccharide (O12) requires an ABC 2 transporter

A recombinant clone encoding enzymes for Klebsiella pneumoniae O12-antigen lipopolysaccharide (LPS) was found when we screened for serum resistance of a cosmid-based genomic library of K. pneumoniae KT776 (O12:K80) introduced into Escherichia coli DH5alpha. A total of eight open reading frames (ORFs) (wb(O12) gene cluster) were necessary to produce K. pneumoniae O12-antigen LPS in E. coli K-12. A complete analysis of the K. pneumoniae wb(O12) cluster revealed an interesting coincidence with the wb(O4) cluster of Serratia marcescens from ORF5 to ORF8 (or WbbL to WbbA). This prompted us to generate mutants of K. pneumoniae strain KT776 (O12) and to study complementation between the two enterobacterial wb clusters using mutants of S. marcescens N28b (O4) obtained previously. Both wb gene clusters are examples of ABC 2 transporter-dependent pathways for O-antigen heteropolysaccharides. The wzm-wzt genes and the wbbA or wbbB genes were not interchangeable between the two gene clusters despite their high level of similarity. However, introduction of three cognate genes (wzm-wzt-wbbA or wzm-wzt-wbbB) into mutants unable to produce O antigen allowed production of the specific O antigen. The K. pneumoniae O12 WbbL protein performs the same function as WbbL from S. marcescens O4 in either the S. marcescens O4 or E. coli K-12 genetic background.     

Behavior of a hybrid F' ts114 lac+, his+ factor (F42-400) in Klebsiella pneumoniae M5a1.

Episome F' ts114 lac+, his+ (F42-400) was transferred from Salmonella typhimurium to Klebsiella pneumoniae. From the progeny, a strain of K. pneumoniae able to retransfer the episome was obtained. The His+ phenotype in this strain is temperature sensitive. Escherichia coli female-specific phages phiII, W31, and T3 were shown to plate on K. pneumoniae. From phiII we obtained two derivatives; phiIIK, which plates only on K. pneumoniae, and phiIIE, which plates only on E. coli. Growth of phages T3 and phiIIK was inhibited by F42-400 in K. pneumoniae. Growth in presence of acridine orange in a defined medium at 40 C resulted in a high level of curing. The frequency of His+ cells after growth in acridine orange at 40 C was 0.001%. An extensive search to detect chromosome mobilization by F42-400 in K. pneumoniae, under different experimental conditions, was negative. We cannot exclude the possibility that the low transfer efficiencies prevented our detection of chromosome mobilization. A search among temperature-resistant, acridine orange-curing-resistant, or galactose-resistant derivatives of the K. pneumoniae donor strain failed to reveal any chromosome transfer. Our failure to detect Hfr's may be a result of: (i) the peculiarity of episome F42-400, (ii) the peculiarity of K. pneumoniae chromosome, or (iii) low transfer efficiency. K. pneumoniae-modified F42-400 and phage 424 were restricted by E. Coli K-12. E. coli K-12-modified episome F42-400 and phage 424 were restricted by K. pneumoniae. E. coli C failed to restrict F42-400 modified with K. pneumoniae specificity. The ability of K. pneumoniae to accept F42-400 is less, by about a factor of 50, than that of E. coli C. As an explanation for the differences in the behavior of E. coli C and K. pneumoniae in ability to receive F42-400 it was suggested that recipient bacteria have specific sites for interaction with the F-pilus tip; these are present in E. Coli C, leading to high transfer efficiency, whereas they may not be present (or if present, are not accessible) in K. pneumoniae, leading to low transfer efficiency.     

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.