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PubMed1985

First strains of the genus Kluyvera in Czechoslovakia.

Fourteen of 21 strains isolated from stools, urine specimens and the hospital environment were identified as Kluyvera. All of these 14 strains corresponded with the literary description of the genus Kluyvera and were identical with two reference strains except that one of them failed to utilize sodium acetate within 7 days. One strain (No. 23441) produced massive growth on Jordan's tartrate, which some Kluyvera do. Important in differentiating indole- and Simmons' citrate-negative Kluyvera strains from Escherichia vulneris (two other of our 21 strains) is negative ornithin decarboxylase and negative Christensen citrate in the latter. Three strains were identified as Citrobacter, where especially indole-positive and urease-negative strains may be reminiscent of Kluyvera. An aberrant strain, No. 25115, which alone failed to grow at 42 degrees C and by some characters differed from Kluyvera, E. vulneris and Enteric Group 10, was identified as E. coli.

Acetates↗
PubMed2003

Antimicrobial resistance in Gram-negative bacilli isolated from infant formulas.

A total of 90 samples of infant formula (IF) were collected from the lactary of a teaching hospital, during a 4-month period from July to August 1999. The sanitary conditions of the formulas were analyzed, and a physiological characterization of Gram-negative bacillus isolates and antimicrobial susceptibility testing were performed. Colony counts were considered to be unacceptable for the majority of the IF samples and the contamination rates were related to inadequate handling. Coliforms (35 degrees C and 45 degrees C growth) were detected in most of the IF tested. Klebsiella pneumoniae, Citrobacter freundii, Cedacea davisae, Klebsiella planticola and Enterobacter cloacae were the isolates most commonly identified. Antimicrobial susceptibility testing showed significant resistance rates, particularly to amoxicillin/clavulanic acid, cefoxitin, cephalotin or ampicillin. One extended-spectrum beta-lactamase-producing K. pneumoniae strain was also recovered.

Amoxicillin-Potassium Clavulanate Combination↗
PubMed1997

Inducible amp C beta-lactamase producing gram-negative bacilli from blood stream infections: frequency, antimicrobial susceptibility, and molecular epidemiology in a national surveillance program (SCOPE).

A surveillance study of nosocomial blood stream infections [Surveillance and Control of Pathogens of Epidemiologic Importance (SCOPE)] was conducted during a 14-month period in 1995 to 1996 in approximately 50 American medical centers. Among the 4725 blood stream infections, the etiologic agent was Enterobacter spp. in 230, Citrobacter freundii in 24, and Serratia marcescens in 65. The vast majority of these isolates (89%) had been sent to the University of Iowa including 198 Enterobacter spp. (46 Enterobacter aerogenes, 141 Enterobacter cloacae, 11 other Enterobacter spp.), 23 C. freundii, and 62 S. marcescens. Because these species are capable of producing Amp C beta-lactamase, we examined their susceptibility to 12 broad-spectrum antimicrobial agents. The frequency of resistance to ceftazidime and the molecular epidemiology of ceftazidime-resistant strains was also examined. Among the Enterobacter spp. and C. freundii isolates, resistance to third generation cephalosporins (ceftazidime, ceftriaxone) and broad-spectrum semisynthetic penicillins (piperacillin), with or without an enzyme inhibitor (piperacillin/tazobactam), was high, e.g., 35 to 50%. The S. marcescens isolates were quite susceptible to all agents tested. Both imipenem and cefepime were active against virtually all isolates tested including 84 stably derepressed Amp C-producing ceftazidime-resistant strains of Enterobacter spp. and C. freundii. The overall rank order of activity for the six best agents against these Amp C-producing strains was: imipenem (100% susceptible) > amikacin = cefepime (98.6%) > ciprofloxacin = gentamicin = ofloxacin (93.6 to 94.0%). Molecular typing studies of ceftazidime-resistant E. cloacae using an automated ribotyping system, as well as pulsed-field gel electrophoresis, indicated that although clonal spread of a single strain occurred in some of the medical centers, most of the episodes of bacteremia were caused by patient-unique strains. Control of these resistant organisms will require attention to microbiologic recognition of phenotypes, to infection control practices, and to limiting the overuse of certain extended spectrum beta-lactams.

Anti-Bacterial Agents↗
PubMed2002

Host/pathogen interactions at mucosal surfaces: immune consequences.

The mucosal immune system has evolved to protect the host against the establishment of infections at or through the mucosal surfaces of the body. Protective immunity must be activated to specific pathogenic agents or their products but inappropriate immune responses to food/environmental antigens must be avoided. Thus, the mucosal immune system is under tight regulation. Pathogenic bacteria and their products can be exploited as specific probes of mucosal immune responses. Bacterial enterotoxins such as cholera toxin are potent mucosal immunogens and adjuvants that activate both mucosal and systemic immune responses. Infection models involving microorganisms such as Citrobacter rodentium can also be used to investigate the consequences of mucosal colonisation that lead to immune disfunction.

Animals↗
PubMed2000

In vitro efficacy of six cephalosporins tested against Enterobacteriaceae isolated at 38 North American medical centres participating in the SENTRY Antimicrobial Surveillance Program, 1997-1998.

The SENTRY Antimicrobial Surveillance Program is an ongoing international collaboration that monitors the predominant bacterial and fungal pathogens and antimicrobial susceptibility patterns associated with community-acquired and nosocomial infections. SENTRY data on the current cephalosporin susceptibility patterns (1997-98) of North American isolates of clinically important Enterobacteriaceae were analyzed. Susceptibility to a selection of cephalosporins was assessed at a central laboratory using reference broth microdilution methods and interpretive criteria specified by the National Committee for Clinical Laboratory Standards. The third- and fourth-generation cephalosporins tested demonstrated excellent activity against Escherichia coli and Klebsiella pneumoniae, whereas some of the older agents maintained good efficacy. Extended spectrum beta-lactamases were detected in all regions of the United States and Canada (1.8-10.7%). Cefepime was the most active agent tested against pathogens with the potential for enzyme-mediated resistance due to Amp C. The third-generation agents maintained acceptable efficacy against Serratia marcescens, but were less effective against Citrobacter and Enterobacter species. The older cephalosporins were generally inadequate against these pathogens, in contrast to cefepime, which was the widest spectrum cephalosporin overall. Some significant regional variations in spectrum were detected.

Cefepime↗
PubMed2003

Enhancement of plasmid curing by 9-aminoacridine and two phenothiazines in the presence of proton pump inhibitor 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone.

Plasmid-containing bacteria often cause serious therapeutic failure during the treatment of infectious diseases. The selection of resistant-mutant strains and the transfer of mobile genetic determinants (such as plasmids and transposons) of resistance promote increased antibiotic resistance. In the last 30 years the antiplasmid effect of acridine dyes, ethidium bromide, sodium dodecyl sulphate and phenothiazines was described. The main aim of this study was to test the mechanism of the antiplasmid effect of promethazine and 9-aminoacridine on doxycycline-resistant enteric bacteria. The antiplasmid effects of promethazine and 9-aminoacridine were studied on plasmid elimination of native plasmid DNA and plasmid DNA isolated from drug-treated cells of plasmid-containing Escherichia coli, Citrobacter freundii and Enterobacter cloacae. The effects of some phenothiazines on plasmid profiles of bacterial strains isolated from urinary tract infections were analysed by agarose gel electrophoresis. Various complex of plasmid DNA were identified in the presence of promethazine, trifluoperazine and 9-aminoacridine in the agarose gel electrophoresis. Doxycycline resistance of tested enteric bacteria was the target of "curing" in the presence of promethazine and trifluoperazine. The frequency of elimination of tetracycline resistance was low despite the formation of antiplasmid compounds complex with isolated plasmid DNA. Tetracycline resistance plasmid was isolated and re-transformed. The plasmid curing effects of promethazine, trifluoperazine and 9-aminoacridine were increased in the presence of a trifluoroketone proton pump inhibitor on E. coli K12 LE140 strain in a model experiment. We propose that the inefficient penetration of antiplasmid compounds could be responsible for the weak plasmid-curing effect in some clinical isolates and that membrane active, calmodulin- and proton pump inhibitors may be combined for plasmid curing in antibiotic-resistant bacteria.

Acetone↗
PubMed2001

Enzymatic synthesis of aza-l-tyrosines.

Tyrosine phenol-lyase from Citrobacter freundii synthesizes 2-aza-L-tyrosine and 3-aza-L-tyrosine from 3-hydroxypyridine and 2-hydroxypyridine, respectively, and ammonium pyruvate.

Alanine↗
PubMed2002

Insights into enzyme evolution revealed by the structure of methylaspartate ammonia lyase.

Methylaspartate ammonia lyase (MAL) catalyzes the magnesium-dependent reversible alpha,beta-elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to mesaconic acid. The 1.3 A MAD crystal structure of the dimeric Citrobacter amalonaticus MAL shows that each subunit comprises two domains, one of which adopts the classical TIM barrel fold, with the active site at the C-terminal end of the barrel. Despite very low sequence similarity, the structure of MAL is closely related to those of representative members of the enolase superfamily, indicating that the mechanism of MAL involves the initial abstraction of a proton alpha to the 3-carboxyl of (2S,3S)-3-methylasparic acid to yield an enolic intermediate. This analysis resolves the conflict that had linked MAL to the histidine and phenylalanine ammonia lyase family of enzymes.

Amino Acid Sequence↗
PubMed2000

Bacterial penetration across the blood-brain barrier during the development of neonatal meningitis.

Bacterial pathogens may breach the blood-brain barrier (BBB) and invade the central nervous system through paracellular and/or transcellular mechanisms. Transcellular penetration, e.g., transcytosis across the BBB has been demonstrated for Escherichia coli K1, group B streptococcus, Listeria monocytogenes, Citrobacter freundii and Streptococcus pneumonia strains. Genes contributing to invasion of brain microvascular endothelial cells include E. coli K1 genes ompA, ibeA, ibeB, and yijP. Understanding the mechanisms of bacterial penetration across the BBB may help develop novel approaches to preventing bacterial meningitis.

Animals↗
PubMed2002

Tyrosine residues at the immunoglobulin-C-type lectin inter-domain boundary of intimin are not involved in Tir-binding but implicated in colonisation of the host.

Intimin is an outer membrane adhesion molecule involved in bacterial adhesion to intestinal epithelium by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium. Intimin binds to the translocated intimin receptor, Tir, which is delivered to the plasma membrane of the host cell by a type III protein translocation system. Intimin is also implicated in binding to a host cell-encoded intimin receptor (Hir). The receptor-binding activity of intimin resides within the carboxy terminus 280 amino acids (Int280) of the polypeptide. Structural analysis of this region revealed two immunoglobulin-like domains, the second of which forms a number of contacts with the distal C-type lectin-like module. Specific orientation differences at this inter-domain boundary, which consists of several tyrosine residues, were detected between the crystal and solution structures. In this study, we determined the influence of site-directed mutagenesis of each of four tyrosine residues on intimin-Tir interactions and on intimin-mediated intimate attachment. The mutant intimins were also studied using a variety of in vitro and in vivo infection models. The results show that three of the four Tyr, although not essential for A/E lesion formation in vitro, are required for efficient colonisation of the mouse host following oral challenge.

Adhesins, Bacterial↗
PubMed1985

Rapid and automated detection of salmonella by electrical measurements.

A rapid method for determining the presence of salmonella in food is described. It consists of pre-enrichment in buffered peptone water modified by the addition of dulcitol and trimethylamine oxide, followed by selective enrichment in a selenite-cystine broth with similar modifications. Changes in the conductance of the selective enrichment broth are monitored continuously using a suitable impediometric instrument. Most of the Salmonella spp. tested gave a fast (approximately 100 microS/h) and large (greater than 600 microS) change in conductance, other enteric bacteria much less or no change. The assay is usually complete within 24 h. Samples of foodstuffs, naturally and artificially contaminated with Salmonella spp., were all correctly classified. Some strains of Citrobacter freundii produced a false positive conductance response, and they could not be selectively eliminated using antibiotics or cyanide. The conductance method is simple and easy to use, gives rapid results and involves less media and subculturing than is required for traditional methods.

Bacteriological Techniques↗
PubMed1987

Inhibition of colonization of the chicken alimentary tract with Salmonella typhimurium gram-negative facultatively anaerobic bacteria.

Oral administration of strains of food poisoning salmonellas to day-old chickens produced a profound inhibition in the subsequent colonization of the caeca by a strain of Salmonella typhimurium given one day later. Closely related genera were unable to produce a similar inhibition. The inhibition was not the result of bacteriophages produced by the first strain. Neither was it the result of an immunological response by the host induced by the first strain. In additional experiments in day-old chickens, inhibition of an Escherichia coli Nalr strain and of a Citrobacter sp. Nalr strain was produced by the antibiotic-sensitive forms of the homologous strains while strains from other genera did not produce any inhibition. When an avirulent mutant of S. typhimurium was used for pre-treatment a statistically significant reduction in the excretion of the super-infecting S. typhimurium Nalr strain over several weeks was produced. A genus specific inhibition was reproduced in vitro by mixed culture experiments. Live cultures were necessary for in vitro inhibition. Killed cells or a culture supernatant produced no inhibition.

Animals↗
PubMed2001

UV Raman spectral intensities of E. coli and other bacteria excited at 228.9, 244.0, and 248.2 nm.

Resonance Raman spectral intensities per average bacterial cell have been measured quantitatively for Gram-negative Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes, as well as Gram-positive Bacillus subtilis and Staphylococcus epidermidis. Spectra have been obtained from cultures in the lag, log, and stationary growth phases excited in turn by 228.9, 244.0, and 248.2 nm light. Although Raman spectral peak positions (cm(-1)) excited by a given wavelength are very similar for all five bacterial species, the organisms are characterized by significantly different spectral intensity values. Intensity changes are associated with growth phase changes in all of the species as well. A comparison of measured with estimated average intensities has been made for spectra of log-phase E. coli. It is possible to compare measured intensities with intensities estimated for log-phase E. coli on the basis of the knowledge of its known average cellular molecular composition. A significant degree of hypochromism is observed in E. coli nucleic acid spectra. In contrast, strong average hyperchromism characterizes all aromatic amino acid peaks belonging to the same E. coli cells. Results suggest that knowledge of spectral intensity values will enhance significantly the capability to identify bacteria by means of their UV resonance Raman spectra.

Bacillus subtilis↗
PubMed1976

Reductive microbial conversion of anthracycline antibiotics.

Reductive conversion of several anthracycline glycosides to their 7-deoxyaglycones occurs during their microaerophilic incubation with strains of Aeromonas hydrophila, Citrobacter freundii, and Escherichia coli. Further, extracts of microaerophilically grown A. hydrophilia catalyze DPNH-dependent reductive conversion of the same compounds. Anthracycline substrates cleaved by both whole cells and by the cell-free system include steffimycin, steffimycin B, nogalamycin, cinerubin A, and daunomycin. Investigation of glycoside cleavage as a function of both time and anthracycline concentration demonstrated the superiority of A. hydrophila over C. freundii and E. coli in regard to reaction rate and efficiency of conversion. Interestingly, some degree of anaerobicity was required for glycoside cleavage by all three organisms. Evidence supporting 7-deoxyaglycone formation via direct reductive cleavage, as opposed to a multienzyme-catalyzed process involving hydrolysis followed by dehydration and reduction, includes the following. Equilibrium mixtures of glycoside substrate and 7-deoxyaglycone product prepared using both whole cells and their extracts display no anthracycline hydrolysis products. Further, authentic steffimycinone (aglycone), the expected product of hydrolytic sugar cleavage of steffimycin, was shown to be converted to 7-deoxysteffimycinone (7-deoxyaglycone) at a rate slower than steffimycin. These data indicate that, if steffimycinone were present as an unbound metabolic intermediate, it should have been visible in the equilibrium mixture, but none was detected.

Aerobiosis↗
PubMed2004

Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism.

Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants, and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) in most bacteria. Here, we compare the PTS-dependent kinase of Escherichia coli and the ATP-dependent kinase of Citrobacter freundii. They display 30% sequence identity. The binding constants of the E. coli kinase for eleven short-chain carbonyl compounds were determined by acetone precipitation of the enzyme-substrate complexes. They are 3.4 microM for Dha, 780 microM for Dha-phosphate (DhaP), 50 microM for D,L-glyceraldehyde (GA), and 90 microM for D,L-glyceraldehyde-3-phosphate. The k(cat) for Dha of the PTS-dependent kinase is 290 min(-1), and that of the ATP-dependent kinase is 1050 min(-1). The Km for Dha of both kinases is <6 microM. The X-ray structures of the enzyme-GA and the enzyme-DhaP complex show that substrates as well as products are bound in hemiaminal linkage to an active-site histidine. Quantum-mechanical calculations offer no indication for activation of the reacting hydroxyl group by the formation of the hemiaminal. However, the formation of the hemiaminal bond allows selection for short-chain carbonyl compounds and discrimination against structurally similar polyols. The Dha kinase remains fully active in the presence of 2 M glycerol, and phosphorylates trace impurities of carbonyl compounds present in glycerol.

Acetone↗
PubMed2006

Structures of apo- and holo-tyrosine phenol-lyase reveal a catalytically critical closed conformation and suggest a mechanism for activation by K+ ions.

Tyrosine phenol-lyase, a tetrameric pyridoxal 5'-phosphate dependent enzyme, catalyzes the reversible hydrolytic cleavage of L-tyrosine to phenol and ammonium pyruvate. Here we describe the crystal structure of the Citrobacter freundii holoenzyme at 1.9 A resolution. The structure reveals a network of protein interactions with the cofactor, pyridoxal 5'-phosphate, and details of coordination of the catalytically important K+ ion. We also present the structure of the apoenzyme at 1.85 A resolution. Both structures were determined using crystals grown at pH 8.0, which is close to the pH of the maximal enzymatic activity (8.2). Comparison of the apoenzyme structure with the one previously determined at pH 6.0 reveals significant differences. The data suggest that the decrease of the enzymatic activity at pH 6.0 may be caused by conformational changes in the active site residues Tyr71, Tyr291, and Arg381 and in the monovalent cation binding residue Glu69. Moreover, at pH 8.0 we observe two different active site conformations: open, which was characterized before, and closed, which is observed for the first time in beta-eliminating lyases. In the closed conformation a significant part of the small domain undergoes an extraordinary motion of up to 12 A toward the large domain, closing the active site cleft and bringing the catalytically important Arg381 and Phe448 into the active site. The closed conformation allows rationalization of the results of previous mutational studies and suggests that the observed active site closure is critical for the course of the enzymatic reaction and for the enzyme's specificity toward its physiological substrate. Finally, the closed conformation allows us to model keto(imino)quinonoid, the key transition intermediate.

Apoenzymes↗
PubMedDate not supplied

Influence of sulfobetaines on the stability of the Citrobacterdiversus ULA-27 beta-lactamase.

The activity and stability of beta-lactamase from Citrobacter diversus ULA-27 have been investigated in the presence of different ionic and zwitterionic surfactants. All the sulfobetaine surfactants tested allow the enzyme to retain its full activity, but the best stabilizing effect is greatly dependent on their structure. Very little variations on the monomer headgroup can significantly reduce enzyme deactivation or speed up the loss of activity with respect to buffer alone. The whole hydrophobic/hydrophilic balance on the headgroup seems to have a determining role in preserving beta-lactamase activity and structure. The presence of zwitterionic surfactants stabilizes the protein conformation toward denaturation by urea and low-temperature inactivation. Similar experiments were performed in the presence of other two zwitterionic surfactants, an amine oxide, dimethylmyristylamine oxide (DMMAO) and a carboxybetaine, cetyldimethylammonium methanecarboxylate (CB1-16). The former stabilizes the enzyme even better than the sulfobetaines, the latter quickly deactivates it. Therefore, the factors responsible for beta-lactamase stabilization are dependent not only on the zwitterionic nature of the surfactant headgroup but also specific interactions between the surfactant and the protein may be important.

Betaine↗
PubMed1996

Design, synthesis, and evaluation of 2 beta-alkenyl penam sulfone acids as inhibitors of beta-lactamases.

A general method for synthesis of 2 beta-alkenyl penam sulfones has been developed. The new compounds inhibited most of the common types of beta-lactamase. The level of activity depended very strongly on the nature of the substituent in the 2 beta-alkenyl group. The inhibited species formed with the beta-lactamase from Citrobacter freundii 1205 was sufficiently stable for X-ray crystallographic studies. These, together with UV absorption spectroscopy and studies of chemical degradation, suggested a novel reaction mechanism for the new inhibitors that might account for their broad spectrum of action. The (Z)-2 beta-acrylonitrile penam sulfone Ro 48-1220 was the most active inhibitor from this class of compound. The inhibitor enhanced the action of, for example, ceftriaxone against a broad selection of organisms producing beta-lactamases. The organisms included strains of Enterobacteriaceae that produce cephalosporinases, which is an exceptional activity for penam sulfones.

Ceftriaxone↗