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Isolation of Chromobacterium spp. from foods, soil, and water.

Chromobacterium violaceum, a soil and water inhabitant, has been implicated in human disease with a high mortality rate, particularly in the southeastern United States. The psychrotrophic Chromobacterium lividum has been isolated from foods, water, and soil, but is not considered pathogenic. To determine the distribution of Chromobacterium spp. in soil, water, and foods in the Gainesville area, we evaluated Bennett, Ryalls and Moss, and Aeromonas membrane agars for their ability to recover these organisms from various samples when incubated at 25 or 35 degrees C. Bennett agar was best for the isolation of both species when incubated at 25 degrees C; however, at 35 degrees C, Aeromonas membrane agar gave the highest recoveries of C. violaceum. C. violaceum was recovered only from soil and water, whereas C. lividum was frequently recovered from foods as well as soil and water.

Chromobacterium

Metabolism of N-carbobenzoxyl-L-tryptophan by Chromobacterium violaceum.

Chromobacterium violaceum (ATCC 12472) metabolizes N-carbobenzoxyl-L-tryptophan into its 2', 3'-dehydro-derivative, and indole-3-propionic acid into indole-3-acrylic acid. The biotransformation accurs in resting cell incubations, and in growing cultures of the bacterium. Tryptophan in fermentation media enhances the ability of Chromobacterium violaceum to perform the conversion by an undetermined mechanism. The amino acid also prevents stored cultures from losing the ability to accomplish the biotransformation. The reaction apparently requires oxygen, and preliminary experiments suggest that it is catalyzed by a dehydrogenase.

Acrylates

Nonfatal and fatal infections caused by Chromobacterium violaceum.

Human infection caused by Chromobacterium violaceum is rare, but when it occurs it is associated with a high mortality rate. We report the cases of two patients with infection due to Chromobacterium violaceum. One patient presented early in the course of disease and survived after receiving appropriate antibiotic therapy. The other patient presented late in the course of disease and died of fulminant sepsis with abscesses in multiple organs. We discuss the similarity between C. violaceum infection and the septicemic form of melioidosis.

Adult

Effective method for activity assay of lipase from Chromobacterium viscosum.

A method was devised for activity assay of the lipase [triacylglycerol acyl-hydrolase, EC 3.1.1.3] excreted from Chromobacterium viscosum into the culture medium; olive oil emulsified with the aid of Adekatol 45-S-8 (a non-ionic detergent, the ethoxylate of linear sec-alcohols having chain lengths of 10--16 carbon atoms) was used as the substrate. This method was specifically effective for Chromobacterium lipase acitvity assay, and was approximately twice as sensitive as the conventional method, in which polyvinyl alcohol is used for the emulsification of the substrate.

Chromobacterium

DNA:rRNA hybridization studies of Chromobacterium fluviatile.

Hybrids were prepared between 14C-labelled rRNA from each of nine species of Gram-negative bacteria and the DNA of Chromobacterium fluviatile. Two parameters were determined for each hybrid - the T m(e), which is the temperature at which 50% of the hybrid was denatured, and the percentage of rRNA bound under defined stringent conditions. The former gives a measure of the stability of the duplex once formed and the latter probability reflects the amount of the genome involved in the coding of rRNA. These parameters were used as a measure of the relatedness of C. fluviatile to each of the other nine species. The results suggest that C. fluviatile is more closely related to C. violaceum than to any other species tested. The taxonomic relationship of C. fluviatile to other Gram-negative bacteria was assessed by incorporating the results into a database already published by De Ley et al. (1978), and the use of principal components analysis was explored as an alternative way of presenting such data. This analysis confirmed the isolated position of C. fluviatile but, until further isolates are studied, it seems best to retain the species in the genus Chromobacterium.

Chromobacterium

Identification of Chromobacterium violaceum: pigmented and non-pigmented strains.

The classification and, therefore, identification of Chromobacterium violaceum has been based upon its ability to produce a violet pigment. Although the organism may yield non-pigmented variants when subcultured on artificial media, the isolation of non-pigmented strains from pathological tissues or from nature had not been reported. With a method established for the identification of C. violaceum regardless of violet pigmentation, non-pigmented strains were isolated from nature. The presence of non-pigmented strains of C. violaceum in nature is of significance to taxonomy and clinical bacteriology. Pigmentation cannot be held as an essential characteristic of the definition of the genus Chromobacterium and gives credence to the suspicion of Sneath (1960, 1966) that the genus is not a natural one. Non-pigmented strains may have been isolated from clinical material but wrongly identified as belonging to other genera of non-pigmented Gram-negative bacilli and regarded as not being pathogenic.

Animal Diseases

Deoxyribonucleic acid homology and taxonomy of Agrobacterium, Rhizobium, and Chromobacterium.

Hybridization experiments were carried out between high molecular weight, denatured, agar-embedded deoxyribonucleic acid (DNA) and homologous, nonembedded, sheared, denatured (14)C-labeled DNA from a strain of Agrobacterium tumefaciens and Rhizobium leguminosarum (the reference strains) in the presence of sheared, nonembedded, nonlabeled DNA (competing DNA) from the same or different nomen-species of Agrobacterium, Rhizobium, Chromobacterium, and several other organisms. Percentage of DNA homology was calculated from the results. The findings are discussed in relation to previous taximetric studies, present classification schemes, and guanine-cytosine content of the DNA. Strains of A. tumefaciens, A. radiobacter, A. rubi, A. rhizogenes, R. leguminosarum, and R. meliloti exhibited a mean percentage of DNA homology greater than 50 with the two reference strains. A. tumefaciens, A. radiobacter, and A. rubi were indistinguishable on the basis of DNA homology, with strain variations for this group involving up to 30% of their base sequences. The remainder of the organisms studied fall into at least six distinct genetic groups: (i) R. (Agrobacterium) rhizogenes, which is more homologous to R. leguminosarum than to the A. tumefaciens-A. radiobacter group; (ii) R. leguminosarum; (iii) R. meliloti; (iv) R. japonicum, which has a mean DNA homology of some 38 to 45% with the reference strains; (v) Chromobacterium, which is as genetically remote from the reference strains as, for example, Pseudomonas; and (vi) A. pseudotsugae strain 180, which has a DNA homology with A. tumefaciens and R. leguminosarum of only about 10%. Since this latter homology value is similar to what was found after hybridizations between the reference strains and organisms such as Escherichia coli and Bacillus subtilis, A. pseudotsugae should definitely be removed from the genus.

Bacillus subtilis

Pathogenicity of nonpigmented cultures of Chromobacterium violaceum.

Nonpigmented cultures of Chromobacterium violaceum have been found to be similar to pigmented cultures in their virulence for mice and the pathology of their infections. Clinicians and microbiologists should be prepared to consider nonpigmented C. violaceum in their differential diagnoses of infections caused by gram-negative bacteria. The laboratorian who is not aware of this possibility is likely to erroneously identify nonpigmented strains of C. violaceum as members of closely associated genera, particularly Aeromonas. It is known that violet pigmentation is not an essential feature or an exclusive character of the genus Chromobacterium. This study has also shown that pigmentation of C. violaceum is not related to its pathogenicity or to the pathology of its infections.

Aeromonas

Fatal Chromobacterium violaceum septicaemia.

A 19-year-old Malay male succumbed to a septicaemia caused by Chromobacterium violaceum 11 days after onset of illness. The organism is a common soil saprophyte and may be considered as contaminant on culture. It is essential to recognise its clinical significance in purulent processes so that appropriate therapy can be instituted. We report the first fatal case of Chromobacterium violaceum infection in the University Hospital, Kuala Lumpur.

Abscess

Chromobacterium violaceum infection in a nonhuman primate (Macaca assamensis).

Chromobacteriosis caused by Chromobacterium violaceum was diagnosed as an Assam macaque, Macaca assamensis, that died 4 days after receipt of the Yerkes Primate Center, It was received from a primate facility in Florida where it has been housed with a group of rhesus monkeys for 5 years. The animal died suddenly without showing any signs of clinical illness. Necropsy findings included extensive hepatic necrosis with the formation of multiple large cavitary lesions. Foci of necrosis were also found in the lungs and lymph nodes. Numerous gram-negative bacilli were demonstrable in the lesions and Chromobacterium violaceum was isolated from the blood, liver, lungs, spleen and kidneys.

Animals

Bioprospecting Chromobacterium violaceum for bioremediation: an alternative to environmental lead pollution.

Lead pollution is a major environmental concern, but current decontamination technologies remain limited due to high costs. Therefore, alternative biotechnological processes have been successfully developed and applied due to their reduced cost and lower aggressiveness in the environment. The remarkable adaptive versatility of Chromobacterium violaceum in metal-contaminated environments makes this bacterium a promising candidate for Pb bioremediation. Therefore, the reference strain C. violaceum ATCC 12,472 and the environmental isolate C. violaceum SCV1, the first strain of this species isolated from a Brazilian area with natural Pb occurrence, were evaluated for Pb resistance under different Pb concentrations and exposure times. Pb biosorption was assessed by scanning electron microscopy, while strain-specific protein profiles were characterized using tandem mass spectrometry-based proteomic analysis. The results obtained revealed the potential of C. violaceum to perform lead bioremediation. Scanning electron microscopy analysis confirmed the biosorption of lead by C. violaceum strains. C. violaceum SCV1 was able to remove up to 40% more lead concentration when compared to ATCC 12,472 which suggested adaptation through natural selection process of C. violaceum SCV1. Proteome analysis revealed 1531 proteins, of which several are candidates for lead bioremediation. This is the first study on the resistance proteomics of C. violaceum against lead. The acclimatization of the bacteria linked to the identification of several proteins related to: biosorption; efflux and ionic uptake (bioaccumulation); biomolecule transport; and biomethylation, point out to this organism as a potential lead bioremediation agent.

Chromobacterium

Chromobacterium violaceum infection of the eye. A report of two cases.

We report what we believe to be the first two known cases of Chromobacterium violaceum infection of the eye. One patient had conjunctivitis as the initial manifestation of an ultimately fatal infection. In the second patient, who suffered from chronic granulomatous disease of childhood, orbital cellulitis developed as part of a disseminated C violaceum infection from which he subsequently recovered. Infections caused by C violaceum may involve the eye and orbit. These infections are frequently fatal, but early recognition and aggressive treatment may result in a higher cure rate.

Bacterial Infections

Kinetics and stability of a Chromobacterium viscosum lipase in reversed micellar and aqueous media.

The lipolytic activity of Chromobacterium viscosum lipase B (EC 3.1.1.3.; triacylglycerol hydrolase) solubilized both in water and AOT/isooctane reversed micelles has been investigated using triolein as a substrate. The influence of relevant parameters in the catalytic activity such as temperature, pH, surfactant and substrate concentrations, and water content was tested and compared in both media. A study of stability of the lipase was carried out, with particular reference to the influence of pH. Three major effects of the encapsulation of the lipase in the micelles were observed: increased activity (up to 7 times higher than in water), greater stability, specially at pH 7, and higher resistance to thermal deactivation.

Chromobacterium

Crystal structure of a bacterial lipase from Chromobacterium viscosum ATCC 6918 refined at 1.6 angstroms resolution.

The crystal structure of a lipase from the bacterium Chromobacterium viscosum ATCC 6918 (CVL) has been determined by isomorphous replacement and refined at 1.6 angstroms resolution to an R-factor of 17.8%. The lipase has the overall topology of an alpha/beta type protein, which was also found for previously determined lipase structures. The catalytic triad of the active center consists of the residues Ser87, Asp263 and His285. These residues are not exposed to the solvent, but a narrow channel connects them with the molecular surface. This conformation is very similar to the previously reported closed conformation of Pseudomonas glumae lipase (PGL), but superposition of the two lipase structures reveals several conformational differences. r.m.s. deviations greater than 2 angstroms are found for the C alpha-atoms of the polypeptide chains from His15 to Asp28, from Leu49 to Ser54 and from Lys128 to Gln158. Compared to the PGL structure in the CVL structure, three alpha-helical fragments are shorter, one beta-strand is longer and an additional antiparallel beta-sheet is found. In contrast to PGL, CVL displays an oxyanion hole, which is stabilized by the amide nitrogen atoms of Leu17 and Gln88, and a cis-peptide bond between Gln291 and Leu292. CVL contains a Ca2+, like the PGL, which is coordinated by four oxygen atoms from the protein and two water molecules.

Amino Acid Sequence

Purification and properties of the extracellular metallo-proteinases of Chromobacterium lividum (NCIB 10926).

Four extracellular proteolytic enzymes (I-IV) (EC 3.4.22.-) were identified in static cultures of Chromobacterium lividum (NCIB 10926) by agar gel electrophoresis and isoelectric focusing. Proteinases I-III were freed of non-enzymic protein by chromatography on TEAE-cellulose and CM-cellulose. The enzyme mixture was then fractionated in a pH gradient by isoelectric focusing. All three enzymes were shown to be heat-labile metallo-enzymes. Optimal activity occurred at pH 5.6 for enzyme I and at pH 6.2 for enzymes II and III. Remazolbrilliant Blue-hide powder was a sensitive substrate for these enzymes. Proteinase I was also shown to degrade haemoglobin and casein effectively, but not myoglobin, ovalbumin or bovine serum albumin. Proteinases I-III exhibited molecular weight values of 75 000, 72 000 and 67 000 by exclusion chromatography and 71 000 and 66 000 by sodium dodecyl sulphate-poly-acrylamide-gel electrophoresis for enzyme I and II, respectively. The amino acid compositions of enzymes I and II were somewhat similar. Proteinase I was inhibited by EDTA, 1,2-di(2-aminoethoxy)ethane-N,N,N',N'-tetraacetic activity. Mg2+ could substitute for Ca2+ or Mn2+ for Co2+. The interrelationship of proteinases I-III is discussed.

Amino Acids

Lipase from Chromobacterium viscosum: biochemical characterization indicating homology to the lipase from Pseudomonas glumae.

Previous purification of a commercial lipolytic preparation from Chromobacterium viscosum using gel filtration chromatography yielded two enzymatically active fractions, named lipases A and B. Characterization of these fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that lipase A consisted of a high molecular weight aggregate of lipase protein with lipopolysaccharides. This complex could be dissociated by treatment with EDTA-Tris buffer containing the non-ionic detergent n-octyl-beta-D-glucopyranoside and subsequent isoelectric focusing in an agarose gel containing the same detergent. Both lipases A and B revealed a major peak corresponding to an isoelectric point of 7.1. SDS-PAGE analysis of lipases A and B after purification by gel filtration or by IEF revealed one major protein band of M(r) of 33 K. Determination of N-terminal amino acid sequences confirmed that both fractions A and B contained the same lipase protein. Furthermore, the N-terminal amino acid sequence of the C. viscosum lipase was identical to the one of Pseudomonas glumae lipase.

Amino Acid Sequence

An acute pleuropneumonia in a pig caused by Chromobacterium violaceum.

A 2.5-month-old, 30 kg Duroc pig died 10 days after showing clinical signs of dyspnoea and diarrhoea. Acute necrotizing and fibrinous pleuropneumonia with locally extensive lesions was found. Chromobacterium violaceum was isolated from pneumonic lung tissues and intratracheal inoculation of a pure culture into two SPF pigs reproduced lesions similar to those found in the natural infection.

Acute Disease

Comparison of the in vitro activity of ciprofloxacin and 24 other antimicrobial agents against clinical strains of Chromobacterium violaceum.

Eleven clinical strains of Chromobacterium violaceum were tested for their susceptibility to 25 antimicrobial agents. Ciprofloxacin was the most active of the compounds tested although norfloxacin and pefloxacin were highly active. No resistance was detected to mezlocillin, piperacillin, apalcillin, imipenem, and aztreonam while a single strain was resistant to ticarcillin. Among the cephalosporin/cephamycin group only cefotetan showed good in vitro activity. Gentamicin was more active than amikacin and tobramycin. Good in vitro activity was also noted for chloramphenicol, doxycycline, and trimethoprim-sulfamethoxazole while C. violaceum strains were highly resistant to rifampin and vancomycin. The bactericidal activity of selected agents was shown to be concentration dependent using time-kill kinetic studies. Addition of clavulanic acid did not increase the activity of ticarcillin and in one case was shown to induce beta-lactamase. High correlation was noted between the broth microdilution and disk diffusion susceptibility tests in predicting the susceptibility patterns of C. violaceum.

Chromobacterium