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Patient cohorting and infection control.

Patients with cystic fibrosis (CF) have an abnormal propensity for recurrent and chronic infections of the lower respiratory tract (LRT), and the most common cause of a shortened lifespan is chronic infection with Pseudomonas aeruginosa. A few other gram-negative organisms, primarily Burkholderia cepacia complex have, however, emerged as serious pathogens capable of establishing chronic LRT infection. Details of these and other CF pathogens can be found in the article by Dr. John LiPuma, Burkholderia and Emerging Pathogens in Cystic Fibrosis, in this issue. Chronically infected patients constitute a major microbial reservoir from which noninfected patients can be infected with both P. aeruginosa and B. cepacia complex by direct patient-to-patient transmission, and possibly also by exposure to contaminated environments. Other more rare pathogens such as Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and nontuberculous mycobacteria (NTM) appear less capable of causing patient-to-patient transmission. Both the physical proximity and the duration of exposure of noninfected patients to patients chronically infected with P. aeruginosa and B. cepacia complex are important determinants of the risk of cross-infection. Cohorting of patients according to presence or absence of specific pathogens coupled with conventional hygienic precautions can, however, lead to a decrease in incidence and prevalence of chronic infections with these two species, wherefore patient cohorting is now an integral component of infection control in patients with CF.

Journal Article↗

Reactions involved in bioluminescence systems of limpet (Latia neritoides) and luminous bacteria.

Luminescence in Latia involves a specific flavoprotein enzyme ("luciferase"), which has a tightly bound flavin group constituting the light-emitter. The overall reaction includes oxidation of a specific substrate ("luciferin," an enol formate derivative of an aliphatic aldehyde), by 2 O(2) molecules, in the presence of a "purple protein" cofactor, yielding a ketone, HCOOH, CO(2), and light. In Achromobacter, a required aliphatic aldehyde, which is functionally equivalent to Latia luciferin, is oxidized to an acid containing the same hydrocarbon chain as the aldehyde; this reaction proceeds in the presence of bacterial luciferase and reduced flavin mononucleotide with a quantum yield of 0.17 + 0.1 photons per aldehyde molecule that is independent of aldehyde chain length from 9 to at least 14 carbons.

Alcaligenes↗

The aldehyde content of luminous bacteria and of an "aldehydeless" dark mutant.

Fatty aldehydes, present in the luminescent cells of Photobacterium phosphoreum and Achromobacter fischeri, and to a very slight extent in the cells of a visually dark, "aldehydeless" mutant of the latter species, were extracted, purified, and oxidized to the corresponding acids. The acids were analyzed by mass spectrometry. The results, in conjunction with various other lines of evidence, indicate that saturated fatty aldehydes, comprising mostly dodecanal, tetradecanal, and hexadecanal, function in the bioluminescent reaction in living cells of these luminous bacteria. The amount of these aldehydes in the cells was computed to be sufficient to sustain steady-state luminescence for a period of about 1 sec, and under such conditions the rate of oxidation of the aldehydes in the process of luminescence must be balanced by their rate of production.

Alcaligenes↗

Amino acid sequence of the von Willebrand factor-binding domain of platelet membrane glycoprotein Ib.

We report the amino acid sequence of a 299-residue segment from the alpha chain of the human platelet membrane glycoprotein Ib. This includes the complete sequence of the amino-terminal tryptic fragment of 290 residues comprising the von Willebrand factor-binding domain. Two primary sets of overlapping fragments were obtained by cleavage of the S-carboxymethylated protein at methionyl and lysyl bonds following treatment with cyanogen bromide and Achromobacter protease I, respectively. Additional fragments were obtained by treatment of native glycocalicin with trypsin, Staphylococcus aureus V8 protease, and Serratia marcescens protease. Analysis of all these fragments provided data that allowed determination of the continuous sequence corresponding to approximately half of the alpha-chain polypeptide. This region of glycoprotein Ib is largely hydrophobic and contains only two N-linked and one O-linked carbohydrate chains. A hydrophilic region exists between residues 215 and 299, which contains a cluster of 10 negatively charged residues at 269-287. This area is likely to attract positively charged molecules. The hydrophilic, highly glycosylated (at serine and threonine residues) region corresponding to the previously described "macroglycopeptide" and representing the carboxyl-terminal half of the alpha chain is likely to begin at residue 292. The determined sequence of the alpha chain of glycoprotein Ib contains a region (residues 29-193) with seven repeats, which is indicative of gene duplication and is highly homologous to human leucine-rich alpha 2-glycoprotein. This protein sequence agrees completely with that deduced from the cDNA sequence reported by Lopez et al. [Lopez, J.A., Chung, D.W., Fujikawa, K., Hagen, F.S., Papayannopoulou, T. & Roth, G.J. (1987) Proc. Natl. Acad. Sci. USA 84, 5615-5619].

Amino Acid Sequence↗

Posttranslationally processed structure of the human platelet protein smg p21B: evidence for geranylgeranylation and carboxyl methylation of the C-terminal cysteine.

smg p21A and -B are small GTP-binding proteins that share putative effector and consensus C-terminal sequences with ras p21 proteins. In the present report, we showed that human platelet smg p21B became labeled when intact platelets were incubated with exogenous [3H]mevalonolactone and when a purified preparation of smg p21B was incubated with bovine brain membranes and S-adenosyl-L-[methyl-3H]methionine. In addition, we demonstrated by gas chromatography/mass spectrometry that treatment of smg p21B with Raney nickel released a geranylgeranyl moiety in a molar ratio of about 1:1. In contrast, treatment of smg p21B with NH2OH or KOH yielded no evidence for the presence of a palmitoyl thioester. Extensive digestion of smg p21B with Achromobacter protease I yielded two C-terminal tripeptides that contained serine and cysteine in a molar ratio of 2:1. Both peptides were modified by a thioether-linked geranylgeranyl group. One of the peptides comigrated with a 3H-labeled proteolytic product of methylated smg p21B on reverse-phase HPLC and this peptide appeared at the same retention time as that of the other peptide after being treated with KOH. Since the cDNA-predicted C-terminal sequence of smg p21B contains a unique Ser-Ser-Cys peptide within its C-terminal domain, -Lys-Lys-Ser-Ser-Cys-Gln-Leu-Leu184, these results indicate that smg p21B is posttranslationally modified by geranylgeranylation of Cys-181 and suggest that further modifications cause proteolytic removal of the three predicted C-terminal amino acids followed by partial methylation of the cysteinyl carboxyl group.

Blood Platelets↗

Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca.

The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented. Sequences of the alpha and beta subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or alpha-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Two-chain botrocetin is a heterodimer composed of the alpha subunit (consisting of 133 amino acid residues) and the beta subunit (consisting of 125 amino acid residues) held together by a disulfide bond. Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the alpha subunit; 2 to 13, 30 to 121, and 98 to 113 of the beta subunit; and 80 of the alpha subunit to 75 of the beta subunit. In terms of amino acid sequence and disulfide bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca(2+)-dependent) lectins.

Amino Acid Sequence↗

Complete amino acid sequence and identification of the platelet glycoprotein Ib-binding site of jararaca GPIb-BP, a snake venom protein isolated from Bothrops jararaca.

Jararaca GPIb-BP, a snake venom protein composed of alpha and beta subunits purified from Bothrops jararaca, binds to platelet glycoprotein (GP)Ib and functions as a receptor blocker for von Willebrand factor binding to GPIb (Fujimura, Y., Ikeda, Y., Miura, S., Yoshida, E., Shima, H., Nishida, S., Suzuki, M., Titani, K., Taniuchi, Y., and Kawasaki, T. (1995) Thromb. Haemostasis 74, 743-750). We present here the entire 142- and 123-residue amino acid sequence of the respective alpha and beta subunits and also demonstrate that the platelet GPIb-binding site resides on the beta and not on the alpha subunit based on an enzyme-linked immunosorbent assay using biotin-labeled jararaca GPIb-BP and competing ligands. Sequences of the alpha and beta subunits were determined by analysis of the intact S-pyridylethylated proteins and their peptides generated by digestion with Achromobacter protease I, Staphyloccocus aureus V8 protease, pepsin, endoproteinase Asp-N, or L-1-tosylamino-2-phenylethyl chloromethyl ketone-trypsin. A 38-39% identity of amino acid sequence between the alpha and beta subunits of jararaca GPIb-BP was observed, as well as a high degree of sequence identities (38-64%) with the respective subunits of botrocetin (Usami, Y., Fujimura, Y., Suzuki, M., Ozeki, Y., Nishio, K., Fukui, H., and Titani, K (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 928-932) and the beta-chain of echicetin (Peng, M., Holt, J. C., and Niewiarowski, S. (1994) Biochem. Biophys. Res. Commun. 205, 68-72).

Amino Acid Sequence↗

Isolation and characterization of rhamnose-binding lectins from eggs of steelhead trout (Oncorhynchus mykiss) homologous to low density lipoprotein receptor superfamily.

Two L-rhamnose-binding lectins named STL1 and STL2 were isolated from eggs of steelhead trout (Oncorhynchus mykiss) by affinity chromatography and ion exchange chromatography. The apparent molecular masses of purified STL1 and STL2 were estimated to be 84 and 68 kDa, respectively, by gel filtration chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry of these lectins revealed that STL1 was composed of noncovalently linked trimer of 31.4-kDa subunits, and STL2 was noncovalently linked trimer of 21.5-kDa subunits. The minimum concentrations of STL1, a major component, and STL2, a minor component, needed to agglutinate rabbit erythrocytes were 9 and 0.2 microg/ml, respectively. The most effective saccharide in the hemagglutination inhibition assay for both STL1 and STL2 was L-rhamnose. Saccharides possessing the same configuration of hydroxyl groups at C2 and C4 as that in L-rhamnose, such as L-arabinose and D-galactose, also inhibited. The amino acid sequence of STL2 was determined by analysis of peptides generated by digestion of the S-carboxamidomethylated protein with Achromobacter protease I or Staphylococcus aureus V8 protease. The STL2 subunit of 195 amino acid residues proved to have a unique polypeptide architecture; that is, it was composed of two tandemly repeated homologous domains (STL2-N and STL2-C) with 52% internal homology. These two domains showed a sequence homology to the subunit (105 amino acid residues) of D-galactoside-specific sea urchin (Anthocidaris crassispina) egg lectin (37% for STL2-N and 46% for STL2-C, respectively). The N terminus of the STL1 subunit was blocked with an acetyl group. However, a partial amino acid sequence of the subunit showed a sequence similarity to STL2. Moreover, STL2 also showed a sequence homology to the ligand binding domain of the vitellogenin receptor. We have also employed surface plasmon resonance biosensor methodology to investigate the interactions between STL2 and major egg yolk proteins from steelhead trout, lipovitellin, and beta'-component, which are known as vitellogenin digests. Interestingly, STL2 showed distinct interactions with both egg yolk proteins. The estimated values for the affinity constant (Ka) of STL2 to lipovitellin and beta' component were 3.44 x 10(6) and 4.99 x 10(6), respectively. These results suggest that the fish egg lectins belong to a new family of animal lectin structurally related to the low density lipoprotein receptor super- family.

Amino Acid Sequence↗

Oxidative modification of tryptophan 43 in the heme vicinity of the F43W/H64L myoglobin mutant.

The F43W/H64L myoglobin mutant was previously constructed to investigate the effects of electron-rich tryptophan residue in the heme vicinity on the catalysis, where we found that Trp-43 in the mutant was oxidatively modified in the reaction with m-chloroperbenzoic acid (mCPBA). To identify the exact structure of the modified tryptophan in this study, the mCPBA-treated F43W/H64L mutant has been digested stepwise with Lys-C achromobacter and trypsin to isolate two oxidation products by preparative fast protein liquid chromatography. The close examinations of the (1)H NMR spectra of peptide fragments reveal that two forms of the modified tryptophan must have 2,6-disubstituted indole substructures. The (13)C NMR analysis suggests that one of the modified tryptophan bears a unique hydroxyl group in stead of the NH(2) group at the amino-terminal. The results together with mass spectrometry (MS)/MS analysis (30 Da increase in mass of Trp-43) indicate that oxidation products of Trp-43 are 2,6-dihydro-2,6-dioxoindole and 2,6-dihydro-2-imino-6-oxoindole derivatives. Our finding is the first example of the oxidation of aromatic carbons by the myoglobin mutant system.

Catalysis↗

Identification of the active site residues of Pseudomonas aeruginosa protease IV. Importance of enzyme activity in autoprocessing and activation.

Protease IV is a lysine-specific endoprotease produced by Pseudomonas aeruginosa whose activity has been correlated with corneal virulence. Comparison of the protease IV amino acid sequence to other bacterial proteases suggested that amino acids His-72, Asp-122, and Ser-198 could form a catalytic triad that is critical for protease IV activity. To test this possibility, site-directed mutations by alanine substitution were introduced into six selected residues including the predicted triad and identical residues located close to the triad. Mutations at any of the amino acids of the predicted catalytic triad or Ser-197 caused a loss of enzymatic activity and absence of the mature form of protease IV. In contrast, mutations at His-116 or Ser-200 resulted in normal processing into the enzymatically active mature form. A purified proenzyme that accumulated in the His-72 mutant was shown in vitro to be susceptible to cleavage by protease IV purified from P. aeruginosa. Furthermore, similarities of protease IV to the lysine-specific endoprotease of Achromobacter lyticus suggested three possible disulfide bonds in protease IV. These results identify the catalytic triad of protease IV, demonstrate that autodigestion is essential for the processing of protease IV into a mature protease, and predict sites essential to enzyme conformation.

Amino Acid Sequence↗

Protein sequence analysis, cloning, and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes. Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation.

Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes, is specifically expressed during the fruiting body formation. We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein. This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis. The N-terminal 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid. The central part of FTX was sequenced with a surface-adhesive 7-kDa fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 M guanidine HCl. The 7-kDa peptide was cleaved with 12 M HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys(251) and Met(252) by trypsin.

Amino Acid Sequence↗

Identity and potential functions of heterotrophic bacterial isolates from a continuous-upflow fixed-bed reactor for denitrification of drinking water with bacterial polyester as source of carbon and electron donor.

A collection of 186 heterotrophic bacteria, isolated directly from a continuous-upflow fixed-bed reactor for the denitrification of drinking water, in which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) granules acted as biofilm carrier, carbon source and electron donor, was studied with regard to taxonomic affiliation and degradation and denitrification characteristics. Two granule samples were taken from a fully operating reactor for enumeration and isolation of heterotrophic bacteria. One sample was drawn from the lower part of the reactor, near the oxic zone, and the other sample from the upper, anoxic part of the fixed bed. Dominant colonies were isolated and the cultures were identified using fatty acid analysis and 16S rDNA sequencing. Their ability to degrade the polymer and 3-hydroxybutyrate and to denitrify in pure culture was assessed. The results show that high numbers of heterotrophic bacteria were present in the biofilms on the polymer granules, with marked differences in taxonomic composition and potential functions between the lower and upper part of the fixed bed. The majority of the isolates were Gram negative bacteria, and most of them were able to reduce nitrate to nitrite or to denitrify, and to utilize 3-hydroxybutyrate as sole source of carbon. Only two groups, one identified as Acidovorax facilis and the other phylogenetically related to Brevundimonas intermedia, could combine denitrification and utilization of poly(3-hydroxybutyrate) (PHB), and were found only in the upper sample. The other groups occurred either in the lower or upper part, or in both samples. They were assigned to Brevundimonas, Pseudomonas, Agrobacterium, Achromobacter, or Phyllobacterium, or were phylogenetically related to Afipia or Stenotrophomonas.

Bacteria↗

Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil.

In rice paddy soils an active cycling of sulfur compounds takes place. To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium. From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate. These strains were characterized by 16S rDNA analyses. The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria. Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp. and Bosea sp. related strains), but five groups represented new S-oxidizers in rice field soil. These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp. or showed sequence similarity to a strain of Achromobacter sp.

Alphaproteobacteria↗

Survey on antibiotic resistance in gram-negative non-fermentative bacteria other than pseudomonas in Italy.

A survey was conducted to investigate the incidence and antibiotic resistance of clinical isolates of Gram-negative non-fermentative bacteria, isolated during a one-year period in Italy. Overall, these microorganisms account respectively for 11.47% and 20.5% of all Gram-negative bacteria. The most representative species isolated during the study were: Acinetobacter, Flavobacterium, Alcaligenes, Achromobacter and Xanthomonas species. As regards their antimicrobial pattern of resistance, this survey demonstrated the wide differences existing within each group. The antimicrobial agents usually active against Pseudomonas aeruginosa do not seem to be useful against these microorganisms.

Ciprofloxacin↗

Catheter-related right-sided endocarditis in bone marrow transplant recipients.

Bone marrow transplant recipients are at increased risk of severe central venous catheter-related septicemias that may be complicated by endocardial infection. In view of this, we prospectively evaluated 141 consecutive patients receiving allogeneic or autologous bone marrow infusion. Seven (5%) of 141 patients developed eight episodes of a clinical syndrome compatible with catheter-related right-sided infective endocarditis; this diagnosis was confirmed at autopsy in two patients who died. Staphylococcus epidermidis was the most frequent isolate (four cases). Other offending pathogens were, in one case each, Enterococcus faecalis, Corynebacterium jeikeium, Pseudomonas alcaligenes, and Achromobacter xylosoxidans plus Candida species. Three- to 7-week courses of antibacterial therapy were associated with a favorable outcome in six of the seven cases. Infective endocarditis may be a complication of the use of central venous catheters and should be actively sought in septicemic bone marrow transplant recipients.

Adolescent↗

Catheter-associated sepsis caused by Ochrobactrum anthropi: report of a case and review of related nonfermentative bacteria.

Ochrobactrum anthropi, formerly known as CDC group Vd, is an oxidase-producing, gram-negative, non-lactose-fermenting bacillus that oxidizes glucose and grows readily on MacConkey agar. Only occasionally isolated from human clinical specimens, this organism has rarely been found to be pathogenic. We describe the first reported case of infection due to O. anthropi in a child, that of bacteremia in a 3-year-old girl undergoing chemotherapy for retinoblastoma. In addition, we review the literature concerning cases of infection due to this and closely related bacterial species, namely Alcaligenes xylosoxidans subspecies xylosoxidans, Agrobacterium radiobacter, and "Achromobacter" group B. Finally, we attempt to clarify the confusing history and taxonomy of these organisms as well as make recommendations regarding antimicrobial therapy for infections caused by them.

Anti-Bacterial Agents↗

Characterization of recombinant murine interleukin 5 expressed in Chinese hamster ovary cells.

We have purified recombinant murine interleukin 5 (rmIL-5) from the supernatant of Chinese hamster ovary cells. Each peptide fragment of the purified rmIL-5 generated by Achromobacter protease I digestion was characterized and glycosylation sites were determined. Although rmIL-5 contains three potential sites of N-linked glycosylation (Asn-26, Asn-55 and Asn-69), Asn-69 is not glycosylated. The oligosaccharides released from the protein by hydrazinolysis were fractionated by paper electrophoresis, lectin column chromatography and gel permeation chromatography, and their structures were analysed by sequential exoglycosidase digestion in combination with methylation analysis. The results indicated that they are a mixture of bi-, tri- and tetraantennary complex-type sugar chains with and without a fucose at the C-6 position of the proximal N-acetylglucosamine residue and high-mannose-type sugar chains. Although > 80% of the sugar chains are neutral oligosaccharides similar to recombinant human IL-5 (rhIL-5; Kodama, S., Endo, T., Tsuroka, N., Tsujimoto, M. and Kobata, A. (1991) J. Biochem., 110, 693-701), rmIL-5 has more tetraantennary oligosaccharides than rhIL-5. A site differential study revealed that Asn-55 has more tetraantennary oligosaccharides than Asn-26.

Amino Acid Sequence↗

In-vitro activity of newer quinolones against aerobic bacteria.

Nalidixic and five newer 4-quinolones, ciprofloxacin, enoxacin, norfloxacin, ofloxacin and pefloxacin were tested against 576 recent clinical aerobic bacterial isolates. The 4-quinolones were regularly active (MIC90 less than 4 mg/l) against the following bacteria: Staphylococcus aureus, S. epidermidis, S. saprophyticus, different Enterobacteriaceae, Haemophilus influenzae, Campylobacter jejuni, Pseudomonas aeruginosa, Agrobacter spp., Aeromonas spp., Plesiomonas spp., Neisseria meningitidis. Other bacteria were usually intermediately susceptible or resistant: different streptococci, Listeria monocytogenes, Nocardia asteroides, P. maltophilia, Achromobacter xylosoxydans and Alcaligenes denitrificans. Ciprofloxacin was the most potent compound, followed by ofloxacin and pefloxacin, norfloxacin and enoxacin being less active. All the 4-quinolones were much more active than nalidixic acid. The MBC/MIC ratios of the 4-quinolones were between 1 and 2 with a majority of strains, and between 2 and 3 with Streptococcus agalactiae, Str. faecalis and L. monocytogenes. A two- to eight-fold increase of MIC was observed by increasing the inoculum 10,000-fold with most of the strains tested. Susceptible bacterial population of Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens and P. aeruginosa contained more clones resistant to nalidixic acid (10(4) to 10(8) at four times the MIC) than to 4-quinolones (10(5) to 10(9) at four times the MIC). Supplementing the media with MgSO4 produced smaller inhibition zone diameters with a disc diffusion method than those obtained with non-supplemented agar, with all quinolone or strains. Less regular effect, or no effect was obtained after supplementation with ZnSO4 or Ca(NO3)2.

Anti-Bacterial Agents↗