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Solubilization and characterization of Chondrosia reniformis sponge collagen.

Chondrosia reniformis sponge collagen, insoluble in its native form, was solubilized by chemical modification of lysyl residues. The solubilized sponge collagen had the same amino acid composition as insoluble collagen and the helicoidal tertiary structure was found by negative Cotton effect to be the same as in native vertebrate collagens. Achromobacter iophagus collagenase, a collagen specific protease, hydrolyzed the soluble sponge collagen. These experiments confirmed that the protein had the same structure as collagen.

Amino Acids↗

Bacterial collagenase and collagen identification.

A method is described for the cleavage of collagenous molecules by bacterial collagenase in the presence of sodium dodecyl sulphate (SDS) and urea. Comparison of three commercially available preparations of bacterial collagenase showed that the most efficient cleavage under these conditions was by the enzyme isolated from Achromobacter iophagus (E.C. 3.4.24.8). No non-specific proteinase activity was apparent in conditions where all collagen types showed some susceptibility to attack. This method represents a simple one-stage identification of collagenous molecules in complex mixtures of proteins and where limited amounts of a protein are available.

Alcaligenes↗

Preparation of Fab' from murine IgG2a for thiol reactive conjugation.

Lysyl endopeptidase (LE) from Achromobacter lyticus M497-1 (EC 3.4.21.50) was utilized to prepare F(ab')2 fragments from mouse anti-P-glycoprotein IgG2a obtained from the UIC2 hybridoma. This report describes a novel single step purification procedure for F(ab')2 fragments that eliminates residual LE activity responsible for secondary cleavage of F(ab')2 to Fab fragments. The purification of F(ab')2 and Fc fragments was accomplished utilizing protein G affinity chromatography and either gradient or step changes in the pH/ionic strength for elution of the Fc and F(ab')2 fragments. Residual LE was eluted from the protein G column with buffer containing 200 mM L-lysine prior to elution of F(ab')2 and Fc fragments. The activity of LE was monitored using the fluorogenic substrate Boc-Val-Leu-Lys-7-amido 4-methyl coumarin. A similar purification procedure for F(ab')2 fragments produced following pepsin digestion of IgG2a is also outlined. The ability of Fab' fragments, from reduced F(ab')2 fragments following LE digestion of IgG2a, to conjugate to thiol reactive groups was demonstrated using N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-meso chlorin e6 mono (N-2-aminoethylamide) (Mce6) conjugates containing reactive maleimide groups. The biological activity of the Fab' targeted HPMA copolymer-Mce6 conjugates was tested against the P-glycoprotein expressing human ovarian carcinoma A2780/AD cell line utilizing a cell survival assay. Fab' targeted HPMA copolymer-Mce6 conjugate demonstrated significantly higher cytotoxicity than either a monoclonal antibody (mAb) targeted HPMA copolymer-Mce6 conjugate or a non-targeted HPMA copolymer-Mce6 conjugate, p < 0.05.

Animals↗

Amoebae-resisting bacteria isolated from human nasal swabs by amoebal coculture.

Amoebae feed on bacteria, and few bacteria can resist their microbicidal ability. Amoebal coculture could therefore be used to selectively grow these amoebae-resisting bacteria (ARB), which may be human pathogens. To isolate new ARB, we performed amoebal coculture from 444 nasal samples. We recovered 7 (1.6%) ARB from 444 nasal swabs, including 4 new species provisionally named Candidatus Roseomonas massiliae, C. Rhizobium massiliae, C. Chryseobacterium massiliae, and C. Amoebinatus massiliae. The remaining isolates were closely related to Methylobacterium extorquens, Bosea vestrii, and Achromobacter xylosoxidans. Thus, amoebal coculture allows the recovery of new bacterial species from heavily contaminated samples and might be a valuable approach for the recovery of as-yet unrecognized emerging pathogens from clinical specimens.

Adult↗

Construction of a leftover bath water model for microbial testing.

In this study, in order to construct a model of leftover bath water, we analyzed one hundred samples of used bath water samples which were provided by twenty-eight volunteer families. It appeared that the number of detected bacteria from such bath water was correlated closely with the number of bathers. Moreover, the pH, acidity, chemical oxygen demand (COD), ion, protein content of the leftover bath water were measured. The number of bathers had no connection with the pH, acidity, COD, and ion content of the leftover bath water. However, the protein content of the bath water correlated with the number of detected bacteria. Based on these results, the model of leftover bath water was constructed. Achromobacter xylosoxidans, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa were incubated with the model bath water as indices of bath water contamination. The number of incubated viable cells in the model bath water increased with increasing concentrations of casamino acid. Consequently, it was suggested that varying the concentration of casamino acid based on family size or contamination would be necessary in the efficient use of the constructed model of leftover bath water for microbial testing.

Baths↗

[Survival of epiphytic microorganisms following sublimation dehydration and storage].

Epiphytic microorganisms belonging to the genera Pseudomonas, Agrobacterium, Achromobacter, Flavobacterium, Mycobacterium, Mycococcus, Nocardia, Corynebacterium, Arthrobacter, Bacillus, and Microbacterium were dehydrated by sublimation employing two suspension media, and were stored at 3-5 degrees C during 1-2.5 years. Survival of epiphytic microorganisms upon dehydration by sublimation and storage was found to depend on their taxonomy (genus, species, strain) and the composition of the suspension medium. The cultures can be stored after dehydration during 2-2.5 years.

Bacteria↗

[Culture and differentiation of obligatory aerobic gram-negative rods from human material; a scheme for application in routine diagnosis (author's transl)].

The diagnosis of obligately aerobic Gram-negative rods in the clinical laboratory may encounter difficulties since media used for Enterobacteriacae are only partially usable for the diagnosis of this group of bacteria (Psuedomonas, Xanthomonas, Alcaligenes, Achromobacter, Brucella, Bordetella, Flavobacterium, Moraxella, Acinetobacter, and some still unnamed taxa). We have developed a diagnostic scheme, based on recent publications in the field and representing an extension of earlier tables from this and other laboratories, which attempts to classify a maximal number of obligately aerobic Gram-negative rods with a minimal number of tests. The scheme, employed on 4051 strains, used blood agar and MacConkey Agar as isolation media. Growth characteristics on these media and microscopic morphology may be of help, but only the type of growth on Triple Sugar Iron (or Kligler's) Agar is characteristic for the group as a whole (no growth in the butt, alkalinization or no pH change on the slant). A primary identification series employs tests for oxidase (Kovacs), oxidation of glucose and xylose (in OF medium), deoxyribonuclease and indole (in DNase Test Agar with Methyl Green), nitrate reduction (in Indole Nitrite Medium), motility (hanging drop), and fluorescein production (on Flo Agar). Results of Kirby-Bauer antimicrobial sensitivity testing serve as additional (colistin) or confirmatory criteria. Incubation is at 30 degrees C for 24-48 hrs. If a diagnosis is not possible than, a secondary series, including tests for lysine decarboxylase (tablets), 4 hr urease, esculin hydrolysis, growth at 42 C and on SS Agar, gelatin liquefaction, and flagellar staining may have to be used, and read after 4-24 hrs at 30 degrees C. Five tables, drawn up according to oxidase, glucose, and xylose reactions, serve to identify the species or taxa. Biotypes cannot be differentiated. The scheme will need updating as more knowledge of these bacteria will become available.

Agar↗

[Products of bacterial destruction of sodium dodecyl sulphate].

The products of Na dodecyl sulphate destruction by the three bacterial cultures--Pseudomonas aeruginosa, Flavobacterium devorans, Achromobacter guttatus--were examined. The cultures were shown to decompose Na dodecyl sulphate in a similar way. The primary mechanism of destruction was found to be hydrolysis of the sulpho-ester bond in the molecule, leading to the separation of sulphate-ion and formation of dodecanol. Products of bacterial destruction of alkyl sulphates did not show foam forming capacity.

Alcaligenes↗

Limited proteolysis of silkworm antitrypsin by several proteinases.

Silkworm antitrypsin (sw-AT) isolated from larval hemolymph was limitedly digested by Achromobacter lysylendopeptidase, alpha-chymotrypsin, subtilisin BPN', subtilisin Carlsberg, papain, or Pseudomonas elastase. Each proteinase could cleave specific site(s) around the reactive site identified for the reaction of sw-AT and bovine trypsin. Among these proteinases, only subtilisin BPN' was inhibited by sw-AT, although weakly. By the cleavable amino acid sequence in sw-AT, it was suggested that whether or not these proteinases were inhibited by sw-AT did not solely depend on their substrate specificities. The susceptibility to the attack of proteinase should indicate that this region is exposed on the molecular surface. The amino acid sequence in the COOH-terminal region slightly away from the reactive site in sw-AT had homology with that in the corresponding region of the serine proteinase inhibitor (serpin) group.

Amino Acid Sequence↗

[Species composition of heterotrophic bacteria in the water of the Rybinek reservoir].

Twenty four cultures of heterotrophic bacteria were isolated from the water of the Rybinsk Reservoir on a medium containing organic matter. Bacteria belonging to the families Pseudomonadaceae and Achromobacteriaceae were most widely distributed; bacteria of the Mycobacteriaceae family were encountered less often. Predominating species which belonged to the genera Pseudomonas, Caulobacter, Flavobacterium, Mycobacterium, and Corynebacterium were isolated from the central part of the Reservoir. The special composition depended on the season. Bacteria of the Caulobacter genus were isolated mainly by the end of July and in May. Bacteria of the Corynebacterium genus predominated in March. Microflora belonging to the genera Pseudomonas and Flavobacterium was more constant. Bacteria isolated on media with the minimum content of organic matter (0.5 mg C per litre) belonged mainly to the Pseudomonas genus. Morphological, cultural, and physiological properties of the bacteria were studied. The bacteria grew on media containing carbohydrates and alcohols as a source of carbon though organic acids were assimilated to a less extent. The activity of catalase was found in the cultures, with an exception of the genera Caulobacter and Achromobacter.

Bacteria↗

[Nosocomial infections in a general surgical ward].

According to the National Nosocomial Infection Surveillance system we analysed the post-surgical nosocomial infections in a surgery ward of Perugia University. Between May 2000 and April 2001, 677 patients were enrolled mean age 51.5 years: 355 (52%) male, 462 (68%) ASA score 1, "clean" surgery in 355 cases (52%), cephazolin prophylaxis in 256 (38%); 11 (2%) patients deceased perioperatively. A total of 37 nosocomial infections, in 33 patients, were detected: 18 pneumonia (48.6%), 10 surgical site infections (27%) with 18 isolated: 12 gram-negative (E. coli 3, Acinetobacter baumannii 2, Providencia stuartii 2, Pseudomonas aeruginosa 2, Achromobacter spp. 1, Citrobacter freundii 1, Morganella morgani 1) and 6 gram-positive (Staphylococcus aureus meticillin resistant 3, Enterococcus faecalis 2, Streptococcus salivarius 1); 7 sepsis (19%) due to 7 gram-positive (S. aureus meticillin resistant 4, S. aureus meticillin susceptible 1, Staphylococcus coagulase negative 1, Clostridium spp 1), 2 urinary tract infections (5.4%). Patients without infections and with nosocomial infections spent in hospital 6.3 and 16.6 days respectively. We can image that in one year 53 surgical procedure were lost, with a lost gain of 79.500-291.500 euro/year.

Bacterial Infections↗

Bacterial arthritis.

The 1991 literature on septic arthritis included a concise review of adult septic arthritis, examples of pseudoseptic arthritis, and two interesting animal studies. One animal study examined the induction of acute synovitis by the intra-articular injection of bacterial endotoxin and the cytokines tumor necrosis factor-alpha, and interleukin-1 beta; and the other studied the effects of early and delayed synovectomy in the management of septic arthritis. The predispositions to septic arthritis can be divided into local joint abnormalities, systemic factors, or both. Examples of the local joint abnormalities include osteoarthritis of the hip and apatite-associated arthropathy. Septic arthritis in a patient with rheumatoid arthritis, in a patient with diabetes mellitus and hip arthropathy associated with hemochromatosis, or in a patient with acquired immunodeficiency syndrome and hemophilic arthropathy are examples of how systemic predisposition is coupled with local joint pathology to increase the vulnerability of the host to joint infection. Other examples of systemic disease that predispose to septic arthritis are systemic lupus erythematosus, hypogammaglobulinemia, and human immunodeficiency virus infection, as well as intravenous drug abuse. Unusual microorganisms causing septic arthritis in the adult include Achromobacter xylosoxidans, Moraxella catarrhalis, meningococci, and diphtheroids. Uncommon pathogenesis is represented by a case of intra-articular inoculation of Mycobacterium gastri into the small joint of the hand and a case of mixed bacterial infection of the hip resulting from an extension of a contiguous pelvic infection associated with trauma. Two cases of immune complex glomerulonephritis illustrate the extra-articular complications of septic arthritis: one due to group G streptococcus and the other due to pneumococcus. Finally, septic bursitis is reviewed from the community practice perspective.

Animals↗

Nucleotide sequence and expression of the ncr nickel and cobalt resistance in Hafnia alvei 5-5.

The structural genes for the nickel and cobalt resistance of the conjugative plasmid pEJH 501 of Hafnia alvei 5-5, contained on a SalI-EcoRI fragment of 4.8 kb, were cloned and sequenced. The DNA sequence included five genes in the following order: ncrA, ncrB, ncrC, ncrY, and ncrX. The predicted amino acid sequences of ncrA were homologous to the amino acid sequences of nreB of Achromobacter xylosoxidans 31A. Expression of ncr with the T7 RNA polymerase-promoter system allowed Escherichia coli BL21 (DE3) to overexpress NcrA, NcrB, and NcrC but not NcrY, and NcrX. The apparent molecular masses of NcrA, NcrB, and NcrC were 30, 33, and 17 kDa, respectively. Primer-extension analysis showed that ncr mRNA started at nucleotide position 23 upstream from ncrA. The promoter region of the ncr operon possessed a strong, putative -35 element of sigma(32)-type promoter sequence, and transcriptional 'lacZ fusion studies indicated that the -35 element influenced sigma(32)-specific transcription.

Base Sequence↗

IAL-CHS (internal airlift loop--ceramic honeycomb supports) reactor used for biodegradation of 2,4-dichlorophenol and phenol.

The internal airlift loop reactor with ceramic honeycomb supports (IAL-CHS) was applied for biodegradation of 2,4-dichlorophenol (2,4-DCP) and phenol. A strain of DCP-degrading bacteria isolated from activated sludge, Achromobacter sp., was rapidly immobilized onto the ceramic honeycomb supports. The immobilized cells effectively biodegraded 2,4-DCP alone and together with phenol in batch and continuous-flow experiments. For example, 2,4-DCP was biodegraded from an influent concentration of 50 mg/L to less than 1 mg/L with a 6-h hydraulic retention time (HRT) in continuous flow tests. The immobilized biomass grew and accumulated through 2,4-DCP biodegradation, and the rate of degradation increased accordingly.

Anthelmintics↗

Technical aspects of gel-based proteomics designed for elucidating an aryl hydrocarbon receptor complex.

The identification of proteins by mass spectrometry has revolutionalized the basic method of identifying proteins constituting an intracellular unit or network for certain biological functions. The gel-based strategy following immunoprecipitation was applied to elucidating proteins associated with the aryl hydrocarbon receptor (AhR). Two hundred femtomoles of AhR was recovered from approximately 2 x 10(7) HepG2 cells by immunoprecipitation and was sufficient for identification by peptide mass fingerprinting. Possible candidates for the AhR-associated proteins were also identified. Improvements of the current strategy to increase the overall sensitivity tenfold are required to clarify the AhR complex in full detail. For example, a combination of trypsin and Achromobacter protease I for in-gel digestion allows the number of missed cleavage sites to be set at zero for database searching, thereby reducing random matches and facilitating identification. There is also room for improvement in each step of sample preparation prior to mass spectrometry.

Cell Line↗

[Application of molecular systematics to study of bacterial cultures consuming volatile organic compounds].

A range of species of four mixed bacterial cultures was studied by molecular systematics methods with the use of 16S rRNA genes. The cultures had been developed for application in minireactors, to degrade volatile organic compounds (VOCs): ethyl benzene, m-xylene, styrene, and o-xylene. A sample of 30 plasmid rDNA clones was obtained for each of the mixed cultures. The clones were analyzed by RFLP according to two restriction sites. Major variants of the 16S-rDNA sequences, corresponding to the most abundant species, were determined for each association. Sequencing of four clones of predominant 16S-rDNAs showed that the culture consuming ethyl benzene was dominated by Pseudomonas fluorescens; o-xylene, by Achromobacter xylosoxydans; styrene, by Pseudomonas veronii; and m-xylene, by Delftia acidovorans. Minor components of all four cultures were generally similar. They included species of the genera Sphingobacter, Rhizobium, Mesorhizobium, Pedobacter, and Paenibacillus. Sampling sequencing of genes for 16S rRNA cloned from total genomic DNA allowed quantitative determination of the composition of actual bacterial associations consuming VOCs in minireactors.

Bacteria↗

['In vitro' activity of different antimicrobial agents on Gram-negative nonfermentative bacilli, excluding Pseudomonas aeruginosa and Acinetobacter spp].

Gram-negative nonfermentative bacilli (NFB) are widely spread in the environment. Besides of difficulties for identification, they often have a marked multiresistance to antimicrobial agents, including those active against Pseudomonas aeruginosa. The objective of this study was to evaluate the 'in vitro' activity of different antimicrobial agents on 177 gram-negative nonfermentative bacilli isolates (excluding Pseudomonas aeruginosa and Acinetobacter spp.) isolated from clinical specimens. Minimum inhibitory concentrations (MIC) were determined according to the Mueller Hinton agar dilution method against the following antibacterial agents: ampicillin, piperacillin, piperacillin-tazobactam, sulbactam, cefoperazone, cefoperazone-sulbactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, colistin, gentamicin, amikacin, trimethoprim-sulfamethoxazole, chloramphenicol, erythromycin, rifampin, norfloxacin, ciprofloxacin and minocycline. Seven isolates: Sphingobacterium multivorum (2), Sphingobacteriumspiritivorum (1), Empedobacterbrevis (1), Weeksella virosa (1), Bergeyella zoohelcum (1) and Oligella urethralis (1), were tested for amoxicillin-clavulanic acid and ampicillin-sulbactam susceptibility, and susceptibility to cefoperazone or sulbactam was not determined. Multiresistance was generally found in Stenotrophomonas maltophilia, Burkholderia cepacia, Chryseobacterium spp., Myroides spp., Achromobacter xylosoxidans, and Ochrobactrum anthropi isolates. On the other hand, Pseudomonas stutzeri, Shewanella putrefaciens-algae, Sphingomonas paucimobilis, and Pseudomonas oryzihabitans, Bergeyella zoohelcum, Weeksella virosa and Oligella urethralis were widely susceptible to the antibacterial agents tested. As a result of the wide variation in antimicrobial susceptibility shown by different species, a test on susceptibility to different antibacterial agents is essential in order to select an adequate therapy. The marked multiresistance evidenced by some species, prompts the need to develop new antimicrobial agents active against this group of bacteria and to search for synergistic combinations.

Anti-Bacterial Agents↗

Crystallization and preliminary X-ray crystallographic analysis of PAS factor from Vibrio vulnificus.

Plasmid Achromobacter secretion (PAS) factor is a putative secretion factor that induces the secretion of periplasmic proteins. PAS factor from Vibrio vulnificus was crystallized at 294 K by the hanging drop vapor-diffusion method. It was isolated as a monomer during the purification procedures. The native crystal belongs to the F222 space group with unit cell parameters a=56.1, b=74.4, c=80.0 A, a=b=g=90 degrees. The crystal was soaked in cryoprotectant containing 1 M NaBr for 1 h for MAD phasing. The diffraction limit of the Br-MAD data set was 1.9 A using synchrotron X-ray irradiation at beam line BL-18B at the Photon Factory, Japan.

Bacterial Proteins↗