Aerobic and anaerobic microbiology of surgical-site infection following spinal fusion

The aerobic and anaerobic microbiology of surgical-site infections (SSI) following spinal fusion was retrospectively studied. This was done by reviewing the clinical and microbiological records at the Naval Hospital in Bethesda, Md., from 1980 to 1992. Aspirates of pus from 25 infection sites showed bacterial growth. Aerobic bacteria only were recovered from 9 (36%) specimens, anaerobic bacteria only were recovered from 4 (16%), and mixed aerobic and anaerobic bacteria were recovered from 12 (48%). Sixty isolates were recovered: 38 aerobes (1.5 isolates per specimen) and 22 anaerobes (0.9 isolate per specimen). The predominant aerobes were Escherichia coli (n = 8) and Proteus sp. (n = 7). The predominant anaerobes were Bacteroides fragilis group (n = 9) and Peptostreptococcus sp. (n = 6) isolates. An increase in recovery of E. coli and B. fragilis was noted in patients with bowel or bladder incontinence. This study highlights the polymicrobial nature of SSI and the importance of anaerobic bacteria in SSI following spinal fusion.     

Efficacy of itraconazole in children with Trichophyton tonsurans tinea capitis

The microbiology of needle aspirates from 40 inflamed cervical lymph glands was studied for aerobic and anaerobic bacteria, fungi and mycobacteria. Forty-two bacterial, 11 mycobacterial and six fungal isolates were isolated. Aerobic bacteria only were recovered in 11 (27.5%), anaerobes alone in five (12.5%) and mixed aerobic and anaerobic bacteria in seven (17.5%). Mycobacterium sp. were recovered in 11 (27.5%) and fungi in six (15%). The recovery of anaerobes was associated with dental infection. Eighteen aerobic bacteria were isolated and the predominant ones were Staphylococcus aureus (eight isolates) and group A streptococci (four). Twenty-four anaerobic bacteria were recovered and the predominant ones were: Prevotella sp. (six), Peptostreptococcus sp. (five), Propionibacterium acnes (four) and Fusobacterium sp. (three). These findings demonstrate the role of anaerobic organisms in cervical lymphadenitis and the need to culture aspirated material from the glands for both aerobic and anaerobic microorganisms.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using-glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers. pH 3.5-7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2-4.6 and 7.5-8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 degrees C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 degrees C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 x 10(3) kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

Analytical applications of immobilized enzymes

Several clinical laboratory methods using enzymes as reagents now may utilize immobilized enzymes. These are enzymes attached to solid surfaces by adsorption, covalent binding, cross linking or similar means. Immobilized enzymes are widely used presently in convenient tests for urine glucose and galactose or blood glucose and urea, and in serum glucose or urea determinations by automated methods. Advantages include enhanced stability, enzyme conservation, reuse and economy. Limitations in clinical analysis include the requirement for proper handling of both the immobilized enzyme and the specimen with which it is used. Immobilized enzymes of the future should aid in the new discoveries regarding sequential enzyme-catalyzed reactions in living cells and expand the utility of enzymes as reagents in analytical laboratory science.     

Aerobic and anaerobic bacteriology of cutaneous abscesses in children

Specimens from 209 cutaneous abscesses in children were cultured for aerobic and anaerobic microorganisms. Of these, nine (4%) were sterile and 51 (24%) yielded pure cultures that were predominantly Staphylococcus aureus. The rest of the abscesses yielded growth of two or more aerobic and/or anaerobic organisms. The data were organized according to these anatomic locations: head, neck, trunk, finger, nailbed, hand, leg, buttocks, perirectal, and vulvovaginal areas. Aerobic bacteria only were present in 92 specimens (46%), anaerobes only were isolated in 52 (26%), and mixed aerobic and anaerobic bacteria were present in 56 abscesses (28%). A total of 467 isolates (270 anaerobes and 197 aerobes) were recovered, accounting for 2.3 isolates per specimen (1.3 anaerobes and 1.0 aerobes). The presence of more than one anaerobe per abscess was obtained from the vulvo-vaginal, buttocks, perirectal, finger, nailbed, and head areas. Aerobes were more prevalent in the neck, hand, leg, and trunk areas. The predominant aerobes recovered were: S aureus (89 isolates), alpha- and nonhemolytic streptococci (29), group A beta-hemolytic streptococci (16), Enterobacter (10), and Escherichia coli (8). The predominant anaerobes recovered were anaerobic Gram-positive cocci (79 isolates), Bacteroides sp (116, including 31 B melaninogenicus group and 29 B fragilis group), and Fusobacterium sp (39). Our findings indicate the polymicrobial nature and predominance of anaerobes in cutaneous abscesses in children in perirectal, head, finger, and nailbed areas.     

The role of calcium on the activity of ERcalcistorin/protein-disulfide isomerase and the significance of the C-terminal and its calcium binding. A comparison with mammalian protein-disulfide isomerase

ERcalcistorin/protein-disulfide isomerase (ECaSt/PDI) shows a 55% identity with mammalian protein-disulfide isomerase (PDI) (Lucero, H. A., Lebeche, D., and Kaminer, B. (1994) J. Biol. Chem. 269, 23112-23119) is a high capacity low affinity Ca2+-binding protein and behaves as a Ca2+ storage protein in the ER of a living cell (Lucero, H. A., Lebeche, D., and Kaminer, B. (1998) J. Biol. Chem. 273, 9857-9863). Here we show that recombinant ECaSt/PDI bound 26 mol of Ca2+/mol and a C-terminal truncated mutant bound 14 mol of Ca2+/mol, both with a Kd of 2.8 mM in 50 mM KCl and 5.2 mM in 150 mM KCl. The percentage reduction in Ca2+ binding in the mutant corresponded with the percentage reduction of deleted pairs of acidic residues, postulated low affinity Ca2+-binding sites. 5 mM Ca2+ moderately increased the PDI activity of both ECaSt/PDI and the C-terminal truncated mutant on reduced RNase and insulin. Surprisingly, ECaSt/PDI in the absence of Ca2+ prevented the spontaneous reactivation of reduced bovine pancreatic trypsin inhibitor. In the presence of 1-5 mM Ca2+ (or 10 microM polylysine) ECaSt/PDI augmented the bovine pancreatic trypsin inhibitor reactivation rate. In contrast, the C-terminal truncated ECaSt/PDI augmented rBPTI reactivation in the absence of Ca2+ and 1-5 mM Ca2+ further accelerated the reactivation rate, responses similar to those obtained with mammalian PDI.     

Penicillium marneffei fungemia in an AIDS patient: the first case report in Taiwan

The microbiology of in 55 ear aspirates obtained from 34 children with chronic otorrhea was studied. Aspiration of the middle ear exudate was done immediately following removal of tympanostomy tube (TT). The middle ear aspirates and swab specimens of the external auditory canals were cultured for aerobic and anaerobic bacteria. Sixty-five isolates were recovered only from the middle ears, 73 only from the external ear canals, and 73 were present at both sites. Analysis of the 138 middle ear isolates demonstrated the recovery of aerobic bacteria only in 28 patients (50%), anaerobes only in seven (13%), and both aerobes and anaerobes in 20 (36%). There were 77 aerobic and 61 anaerobic isolates. Commonly recovered aerobes were Pseudomonas aeruginosa (17 isolates), Staphylococcus aureus (11), Proteus sp. (7), Moraxella catarrhalis (6), Klebsiella pneumoniae (5) and non-typable Haemophilus influenzae (5). Commonly isolated anaerobes were Peptostreptococcus sp. (25 isolates), Prevotella sp. (10), Bacteroides sp. (8) and Fusobacterium sp. (6). Pseudomonas aeruginosa and S. aureus were more often isolated in children older then 6 years. These findings demonstrate the polymicrobial bacteriology of TT-related otorrhea in children. Specimens collected from the external auditory canals can be misleading. Reliable information can be obtained from the ear exudes when collected through the TT or through the open perforation after their removal.     

Analysis of the reaction coordinate of photosynthetic water oxidation by kinetic measurements of 355 nm absorption changes at different temperatures in photosystem II preparations suspended in either H2O or D2O

Flash-induced absorption changes at 355 nm were measured at different temperatures within the range of 2 degrees C S2) = 14 kJ/mol, EA(S2-->S3) = 35 kJ/mol, and EA(S3-->-->S0 + O2) = 21 kJ/mol for theta > 11 degrees C, 67 kJ/mol for theta < 11 degrees C in PS II core complexes dissolved in H2O; (b) replacement of exchangeable protons by deuterons causes only minor changes ( S2, S2 --> S3, and S3 -->--> S0 + O2, respectively. The corresponding values of PS II membrane fragments are 1.3, 1.3, and 1. 4. Based on these results and corresponding EA data reported in the literature for PS II membrane fragments from spinach [Renger, G., & Hanssum, B. (1992) FEBS Lett. 299, 28-32] and PS II particles from the thermophilic cyanobacterium Synechococcus vulcanus Copeland [Koike, H., Hanssum, B., Inoue, Y., & Renger, G. (1987) Biochim. Biophys. Acta 893, 524-533], the reaction coordinate of the redox sequence in the WOC is inferred to be almost invariant to the evolutionary development from cyanobacteria to higher plants. Furthermore, the rather high activation energy of the S2 --> S3 transition provides evidence for a significant structural change coupled with this reaction. Implications for the mechanism of photosynthetic water oxidation are discussed.     

Nitrite-specific active transport system of the cyanobacterium Synechococcus sp. strain PCC 7942

Studies on the nitrite uptake capability of a mutant of Synechococcus sp. strain PCC 7942 lacking the ATP-binding cassette-type nitrate-nitrite-bispecific transporter revealed the occurrence of a nitrite-specific active transport system with an apparent Km (NO2-) of about 20 microM. Similar to the nitrate-nitrite-bispecific transporter, the nitrite-specific transporter was reversibly inhibited by ammonium in the medium.     

A serial CT scan and MRI verification of diffuse cerebrospinal gliomatosis: a case report with stereotactic diagnosis and radiological confirmation

The purpose of this work was to study H2/CO2-utilizing acetogenic population in the colons of non-methane-producing individuals harboring low numbers of methanogenic archaea. Among the 50 H2-consuming acetogenic strains isolated from four fecal samples and an in vitro semi-continuous culture enrichment, with H2/CO2 as sole energy source, 20 were chosen for further studies. All isolates were Gram-positive strict anaerobes. Different morphological types were identified, providing evidence of generic diversity. All acetogenic strains characterized used H2/CO2 to form acetate as the sole metabolite, following the stoichiometric equation of reductive acetogenesis. These bacteria were also able to use a variety of organic compounds for growth. The major end product of glucose fermentation was acetate, except for strains of cocci that mainly produced lactate. Yeast extract was not necessary, but was stimulatory for growth and acetogenesis from H2/CO2.     

Immobilization of N-carbamyl-D-amino acid amidohydrolase

N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombinant Escherichia coli cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-amino acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the adsorbed enzyme. The activity of DCase expressed on Duolite A-568 and Chitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-resin, respectively. DCase immobilized on Duolite A-568 was found to be most stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfite. The stability during the repeated batch reactions was greatly improved when dithiothreitol was in the reaction mixture, and the higher crosslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of the immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyde, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixture was 12%, 18%, and 63%, respectively. DCase produced with Pseudomonas sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are thermotolerant soil bacteria, and that with Agrobacterium sp. strain KNK712 were also immobilized on Duolite A-568. The stability of the enzymes of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than that of strain KNK712.     

Microbial O2- and H2O2-electrode sensors for alcohol assays based on the use of permeabilized mutant yeast cells as the sensitive bioelements

Two types of alcohol-specific microbial/electrochemical biosensors have been developed using specially constructed mutant cells of the methylotrophic yeast Hansenula polymorpha. The cells were immobilized in a calcium alginate gel, and placed between two membranes on the surface of oxygen or hydrogen peroxide-electrodes. The O2 electrode based biosensor contained mutant cells with strongly elevated alcohol oxidase activity. The peroxide electrode based biosensor consisted of catalase-defective mutant cells which produce hydrogen peroxide in the presence of alcohol. Both types of mutant cells were used in permeabilized form in order to release some components of the cellular respiration system, thus increasing the selectivity of the cellular respiration response to alcohol (cell/O2-biosensor) Permeabilization also increased sensitivity of the signal and shortened the response time (cell/H2O2-biosensor). Cell/O2 biosensors were linear up to 1.2 mM for ethanol and 0.35 mM for methanol, cell/H2O2 biosensors were linear up to 4.0 mM for ethanol, and 1.2 mM for methanol. Results were reproducible, sample pretreatment was not required, and the sensors exhibited good operational and storage stability. The use of sucrose, dulcitol or inositol during the preparation of the sensors resulted in increased stability of cells during their liophilization and storage in the dried state. Both biosensors had similar selectivity towards alcohols in the order of methanol (100%), ethanol (21%), and formaldehyde (12%). No signal was observed with glucose or glycerol as substrates.     

The alpha/beta subunit interaction in H(+)-ATPase (ATP synthase). An Escherichia coli alpha subunit mutation (Arg-alpha 296-->Cys) restores coupling efficiency to the deleterious beta subunit mutant (Ser-beta 174-->Phe)

The Ser-beta 174 residue of the Escherichia coli H(+)-ATPase beta subunit has been shown to be near the catalytic site together with Gly-beta 149, Gly-beta 172, Glu-beta 192, and Val-beta 198 (Iwamoto, A., Park, M.-Y., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 3156-3160). In this study, we introduced various residues at position 174 and found that the larger the side chain volume of the residue introduced, the lower the enzyme activity became. The Phe-beta 174 mutant was defective in energy coupling between catalysis and transport, whereas the Leu-beta 174 mutant could couple efficiently, although both mutants had essentially the same ATPase activities (approximately 10% of the wild type). The defective energy coupling of the Phe-beta 174 mutant was suppressed by the second mutation (Arg-alpha 296-->Cys) in the alpha subunit. The Cys-alpha 296/Phe-beta 174 mutant had essentially the same membrane ATPase activity as the Phe-beta 174 single mutant when assayed under the conditions that stabilize the double mutant enzyme. These results indicate the importance of the alpha/beta interaction, especially that between the regions near Arg-alpha 296 and Ser-beta 174, for energy coupling in the H(+)-ATPase. The 2 residues (Ser-beta 174 and Arg-alpha 296) may be located nearby at the interface of the two subunits. About 1 mol of N-[14C]ethylmaleimide could bind to 1 mol of the alpha subunit of Cys-alpha 296/Phe-beta 174 or Cys-alpha 296 mutant ATPase, but could not inhibit the enzyme activity. This is the first intersubunit mutation/suppression approach to ATPase catalysis and its energy coupling.     

Increased activity of a new chlorofluoroquinolone, BAY y 3118, compared with activities of ciprofloxacin, sparfloxacin, and other antimicrobial agents against anaerobic bacteria

A total of 435 clinical isolates of anaerobes were tested with a broth microdilution method to determine the activity of BAY y 3118 compared with those of other agents against anaerobic bacteria. All strains of Bacteroides capillosus, Prevotella spp., Porphyromonas spp., Fusobacterium spp., Clostridium spp., Eubacterium spp., Peptostreptococcus spp., and Veillonella parvula were susceptible (MICs of < or = 2 micrograms/ml) to BAY y 3118. Against the 315 strains of the Bacteroides fragilis group, five strains required elevated MICs (> or = 4 micrograms/ml) of BAY y 3118. Only imipenem and metronidazole were active against all anaerobes. Overall, BAY y 3118 was more active than ciprofloxacin, sparfloxacin, piperacillin, cefotaxime, and clindamycin against the test isolates.     

Synthesis of novel cyclic protease inhibitors using Grubbs olefin metathesis

The microbiologic features of infected sinus aspirates in nine children with neurologic impairment were studied. Anaerobic bacteria, always mixed with aerobic and facultative bacteria, were isolated in 6 (67%) aspirates and aerobic bacteria only in 3 (33%). There were 24 bacterial isolates, 12 aerobic or facultative and 12 anaerobic. The predominant aerobic isolates were Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus (2 each) and Proteus mirabilis, Pseudomonas aeruginosa, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae (1 each). The predominant anaerobes were Prevotella sp. (5), Peptostreptococcus sp. (4), Fusobacterium nucleatum (2), and Bacteroides fragilis (1). Beta-lactamase-producing bacteria were isolated from 8 (89%) patients. Organisms similar to those recovered from the sinuses were also isolated from tracheostomy site and gastrostomy wound aspirates in five of seven instances. This study demonstrates the uniqueness of the microbiologic features of sinusitis in neurologically impaired children, in which, in addition to the organisms known to cause infection in children without neurologic impairment, facultative and anaerobic gram-negative organisms that can colonize other body sites are predominant.     

Induction of the heat shock protein ClpB affects cold acclimation in the cyanobacterium Synechococcus sp. strain PCC 7942

The heat shock protein ClpB is essential for acquired thermotolerance in cyanobacteria and eukaryotes and belongs to a diverse group of polypeptides which function as molecular chaperones. In this study we show that ClpB is also strongly induced during moderate cold stress in the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. A fivefold increase in ClpB (92 kDa) content occurred when cells were acclimated to 25 degrees C over 24 h after being shifted from the optimal growth temperature of 37 degrees C. A corresponding increase occurred for the smaller ClpB' (78 kDa), which arises from a second translational start within the clpB gene of prokaryotes. Shifts to more extreme cold (i.e., 20 and 15 degrees C) progressively decreased the level of ClpB induction, presumably due to retardation of protein synthesis within this relatively cold-sensitive strain. Inactivation of clpB in Synechococcus sp. increased the extent of inhibition of photosynthesis upon the shift to 25 degrees C and markedly reduced the mutant's ability to acclimate to the new temperature regime, with a threefold drop in growth rate. Furthermore, around 30% fewer delta clpB cells survived the shift to 25 degrees C after 24 h compared to the wild type, and more of the mutant cells were also arrested during cell division at 25 degrees C, remaining attached after septum formation. Development of a cold thermotolerance assay based on cell survival clearly demonstrated that wild-type cells could acquire substantial resistance to the nonpermissive temperature of 15 degrees C by being pre-exposed to 25 degrees C. The same level of cold thermotolerance, however, occurred in the delta clpB strain, indicating ClpB induction is not necessary for this form of thermal resistance in Synechococcus spp. Overall, our results demonstrate that the induction of ClpB contributes significantly to the acclimation process of cyanobacteria to permissive low temperatures.     

A comparison of gene organization in the zwf region of the genomes of the cyanobacteria Synechococcus sp. PCC 7942 and Anabaena sp. PCC 7120

The region of the genome encoding the glucose-6-phosphate dehydrogenase gene zwf was analysed in a unicellular cyanobacterium, Synechococcus sp. PCC 7942, and a filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7120. Comparison of cyanobacterial zwf sequences revealed the presence of two absolutely conserved cysteine residues which may be implicated in the light/dark control of enzyme activity. The presence in both strains of a gene fbp, encoding fructose-1,6-bisphosphatase, upstream from zwf strongly suggests that the oxidative pentose phosphate pathway in these organisms may function to completely oxidize glucose 6-phosphate to CO2. The amino acid sequence of fructose-1,6-bisphosphatase does not support the idea of its light activation by a thiol/disulfide exchange mechanism. In the case of Anabaena sp. PCC 7120, the tal gene, encoding transaldolase, lies between zwf and fbp.     

Streptococcal protein H forms soluble complement-activating complexes with IgG, but inhibits complement activation by IgG-coated targets

Protein H, a surface protein of Streptococcus pyogenes interacting with the constant Fc region of IgG, is known to be released from the streptococcal surface by a cysteine proteinase produced by the bacteria. Poststreptococcal glomerulonephritis and rheumatic fever are conditions in which immune complexes and autoimmune mechanisms have been suggested to play pathogenetic roles. The present study demonstrates that addition of protein H to human serum produces complement activation with dose-dependent cleavage of C3. The activation was IgG-dependent and the result of complexes formed between IgG and protein H. These complexes were size heterogeneous with molecular masses of 400 kDa to 1.4 MDa. Using complement-depleted serum reconstituted with complement proteins, the activation by protein H was found to be dependent of the classical, but independent of the alternative pathway of complement. In contrast to results of experiments based on soluble protein H.IgG complexes, complement activation was inhibited by protein H when IgG was immobilized on a surface. The interaction between C1q and immunoglobulins represents the first step in the activation of the classical pathway, and protein H efficiently inhibited the binding of C1q to IgG immobilized on polyacrylamide beads. Protein H reduced C3 deposition on the IgG-coated beads and inhibited immune hemolysis of IgG-sensitized erythrocytes. Finally, significantly less C3 was deposited on the surface of protein H-expressing wild-type streptococci than on the surface of isogenic mutant bacteria devoid of protein H. The results demonstrate that protein H.IgG complexes released from the streptococcal surface can produce complement breakdown at the sites of infection, whereas complement activation on bacterial surfaces is inhibited. This should have important implications for host-parasite relationships. In addition, soluble protein H.IgG complexes might contribute to immunological complications of streptococcal infections.     

Bacillus isolates from the spermosphere of peas and dwarf French beans with antifungal activity against Botrytis cinerea and Pythium species

ClpP functions as the proteolytic subunit of the ATP-dependent Clp protease in eubacteria, mammals and plant chloroplasts. We have cloned a clpP gene, designated clpP1, from the cyanobacterium Synechococcus sp. PCC 7942. The monocistronic 591 bp gene codes for a protein 80% similar to one of four putative ClpP proteins in another cyanobacterium, Synechocystis sp. PCC 6803. The constitutive ClpP1 content in Synechococcus cultures was not inducible by high temperatures, but it did rise fivefold with increasing growth light from 50 to 175 micromol photons m(-2) s(-1). A clpP1 inactivation strain (delta clpP1) exhibited slower growth rates, especially at the higher irradiances, and changes in the proportion of the photosynthetic pigments, chlorophyll a and phycocyanin. Many mutant cells (ca. 35%) were also severely elongated, up to 20 times longer than the wild type. The stress phenotype of delta clpP1 when grown at high light was confirmed by the induction of known stress proteins, such as the heat shock protein GroEL and the alternate form of PSII reaction center D1 protein, D1 form 2. ClpP1 content also rose significantly during short-term photoinhibition, but its loss in delta clpP1 did not exacerbate the extent of inactivation of photosynthesis, nor affect the inducible D1 exchange mechanism, indicating ClpP1 is not directly involved in D1 protein turnover.