Collagenolytic Activity of Cured Hide Bacteria

S ummary : The aerobic and anaerobic collagenolytic activities of bacterial populations from 1 Australian and 2 South African cured hide samples at 0·85, 2·34, 7·0 and 10·0% of NaCl are described. The rate of denaturation of pure, undegraded collagen was determined by the release of amino acids and considerable collagenolysis was demonstrated. Collagenolytic activity was highest under aerobic conditions. Australian hides which produce uniformly good leather with little or no decay lacked collagenolytic bacteria.     

Collagenolytic activity of carrageenin granuloma in rats.

Collagenolytic activity at various phases of the development of carrageenin granuloma was investigated by measuring the amount of dialysable hydroxyproline formed during incubation in vitro of minced granulation tissue. Collagenolytic activity reached a maximum at day 8 after carrageenin injection and then decreased gradually, while collagen synthetic activity was rapidly decreased from day 4 to day 11. The significance of dialysable hydroxyproline in native collagen breakdown of carrageenin granuloma is discussed.     

Collagenolytic activity of carrageenin granuloma in rats

Collagenolytic activity at various phases of the development of carrageenin granuloma was investigated by measuring the amount of dialysable hydroxyproline formed during incubation in vitro of minced granulation tissue. Collagenolytic activity reached a maximum at day 8 after carrageenin injection and then decreased gradually, while collagen synthetic activity was rapidly decreased from day 4 to day 11. The significance of dialysable hydroxyproline in native collagen breakdown of carrageenin granuloma is discussed.     

Glycocalyceal bodies in a human rectal carcinoma cell line and their interstitial collagenolytic activities.

In co-cultivation on a membrane of connective tissue matrix (CTM) obtained from human dura mater, human adenocarcinoma cells (RCM-1) degraded CTM. Morphologically, the destruction of CTM was associated with the shedding of membrane vesicles from the cells. Transmission electron microscopy, using ruthenium red (RR), showed that the shed vesicles were composed of various-sized membrane bound vesicles (MV). A large majority were small glycocalyceal bodies (G-bodies) measuring 20-120 nm in diameter, composed of an amorphous matrix of moderate electron-density surrounded by an RR-positive, trilaminar membrane. G-bodies were separated from medium-sized and large MVs by ultracentrifugation. Ultrastructural observation of the isolated collagen fibrils from CTM co-cultured with RCM-1 cells, showed G-bodies attached to degraded collagen fibrils with characteristic transverse notches along their axes. The lesions occurred as microerosions in the apolar region between the e and d bands of collagen fibrils. Collagenolytic activity in serum-free RCM-1 conditioned medium was localized in the G-body and MV fractions (80% and 20% of the total activity, respectively, when tested against 3H-labeled type I collagen). No activity was detected in the supernatant. The activity in G-bodies was also confirmed by ultrastructural analysis using reconstituted native type I collagen fibrils. The results suggest that RCM-1 cells release interstitial collagenase as a component of G-bodies which facilitates local breakdown of connective tissue during the process of invasion.     

The role of encapsulated anaerobic bacteria in synergistic infections

Abstract: The effect of encapsulation on the virulence, survival, and protection of anaerobic bacteria from phagocytosis is reviewed. Support for the importance of encapsulated Gram-negative anaerobic rods ( Bacteroides sp., Prevotella sp. and Porphyromonas sp.), anaerobic and facultative Gram-positive cocci (AFGPC) was provided by their higher recovery rate in oropharyngeal infections, abscesses and blood, compared to their number in the normal flora. The pathogenicity of Bacteroides, Fusobacterium, Clostridium , and AFGPC was studied by inoculating them into mice and observing their ability to induce subcutaneous abscesses. Encapsulated Bacteroides, Fusobacteria , and AFGPC generally induced abscesses, whereas non-encapsulated organisms did not. However, many of the strains that had only a minimal number of encapsulated organisms (< 1%) survived in the abscesses, and they became heavily encapsulated when inoculated with other viable or non-viable encapsulated bacteria. Thereafter, these strains were able to induce abscesses when injected alone. Encapsulated Gram-negative anaerobic rods and AFGPC-induced bacteraemia and translocation, and increased the mortality of the infected animals more often than did the non-encapsulated form of the same strains. As determined by using selective antimicrobial therapy and quantitative cultures of abscesses induced in mice, possession of a capsule generally made Gram-negative anaerobic rods more important than their aerobic counterparts. Synergistic potentials were seen between encapsulated Gram-negative anaerobic rods and all tested aerobic bacteria and most AFGPC, and also between most AFGPC and Pseudomonas aeruginosa or Staphylococcus aureus . These studies demonstrated the importance of encapsulated anaerobes in mixed infections.     

Collagenolytic cathepsin activity in rabbit peritoneal polymorphonuclear leucocyte granules.

Collagenolytic cathepsin activity was detected in lysed rabbit peritoneal polymorphonuclear leucocytes. The pH optimum was around 3, and activity was greatly enhanced by the presence of cysteine and EDTA. Digestion of polymeric collagen resulted in the release of alpha, beta, and gamma-chains. Collagenolytic cathepsin activity was associated mainly with the granule fraction isolated from homogenates by differential centrifugation. The granule fraction was further fractionated by isopycnic density-gradient centrifugation, and the collagenolytic cathepsin activity was shown to be associated with the azurophil and tertiary granules, both lysosome-like organelles.     

Detection of collagenase activity in oral bacteria

Collagenolytic activity of 12 species of oral bacteria was assessed using two methods of detection. Except for two species, all bacterial strains tested were capable of degrading at least one general protein substrate. Results of collagenolytic activity in a growth assay indicate that Bacteroides gingivalis is the only bacterium capable of degrading collagen when the substrate is sterilized using ethylene oxide. However, if the substrate is sterilized by autoclaving, in the presence or absence of the growth medium, other bacterial species could be shown to be collagenolytic. Collagenolytic activity was also demonstrated when whole or broken cells were used in a [14C]collagen assay. Results from this assay and from inhibition studies indicate that collagenolytic activity can either be the result of the combined activities of both a specific collagenase and nonspecific proteases (B. gingivalis) or nonspecific proteases only (other strains in this study), although in the latter case, the time taken to hydrolyze collagen can be 10 times longer than with a specific collagenase.     

Production and consumption of nitric oxide by denitrifying bacteria under anaerobic and aerobic conditions

Abstract: Pseudomonas aeruginosa, P. stutzeri and Azospirillum brasilense showed highest NO production rates and NO consumption rate constants when anaerobically grown cells were tested under anaerobic conditions. Aerobic assay conditions resulted in 20–75-fold lower NO production rates. NO consumption rate constants, however, decreased by less than a factor of four. NO consumption activity was observed even in aerobically grown P. aeruginosa , provided the assay was done under anaerobic conditions. Obviously, NO consumption was less O₂-sensitive than NO production so that compensation between production and consumption occurred at lower NO mixing ratios under aerobic than under anaerobic conditions.     

Bovine spleen cathepsin B1 and collagenolytic cathepsin. A comparative study of the properties of the two enzymes in the degradation of native collagen.

Bovine spleen cathepsin B1 and collagenolytic cathepsin were separated by chromatography on Amberlite IRC-50 and collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50). 2. Collagenolytic cathepsin degraded insoluble tendon collagen maximally at pH 3.5 and 28 degrees C; mainly alpha-chain components were released into solution. At 28 degrees C the telopeptides in soluble skin collagen were also cleaved to yield alpha-chain components. Collagenolytic cathepsin was thus similar to cathepsin B1 in its action against native collagen, but mixtures of these two enzymes exhibited a synergistic effect. 3. The addition of thiol-blocking compounds produced similar inhibition of collagenolytic cathepsin and cathepsin B1. The enzyme responded similarly to all other compounds tested except to 6-aminohexanoic acid, when collagenolytic cathepsin was slightly activated and cathepsin B1 was almost unaffected. 4. Leupeptin, which is a structural analogue of arginine-containing synthetic substrates, inhibited collagenolytic cathepsin as effectively as cathepsin B1. Collagenolytic cathepsin was shown to retain a low residual activity against alpha-N-benzoyl-DL-arginine p-nitroanilide during purification which was equivalent to 0.2% of the activity of cathepsin B1. 5. Cathepsin B1 and collagenolytic cathepsin could not be separated by affinity chromatography on organomercurial-Sepharose 4B. The two enzymes could be resolved on DEAE-Sephadex (A-50) and by isoelectric focusing in an Ampholine pH gradient. The pI of the major cathepsin B1 isoenzyme was 4.9 and the pI of collagenolytic cathepsin was 6.4. 6. From chromatography on Sephadex G-75 (superfine grade) the molecular weights were calculated to be 26000 for cathepsin B1 and 20000 for collagenolytic cathepsin. The difference in molecular weight was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.     

Effect of antimicrobial therapy on bowel flora and bacterial infection in irradiated mice

Mice exposed to 10 Gy cobalt-60 radiation were given intramuscular antimicrobial therapy of gentamicin, or metronidazole, or a combination of the two. Mortality in the mice treated with metronidazole alone or in combination with gentamicin occurred earlier than in the controls (P less than 0.001). Microorganisms were recovered from the blood, spleen, and liver of the metronidazole-treated mice earlier than from other groups. The predominant organisms recovered from these animals were Enterobacteriaceae. Quantitative cultures of the ileal flora showed a decrease in the number of aerobic, facultative anaerobic and strict anaerobic bacteria after irradiation, and a subsequent increase only in the number of strict aerobic bacteria. As compared to untreated mice, a rapid decrease (by 8.8 logs) in the number of anaerobic flora occurred in the mice treated with metronidazole 5 days after irradiation. This was followed by a rapid increase in the number of aerobic organisms which coincided with the earlier mortality in this group. These data suggest that antimicrobial agents that decrease the number of the strict anaerobic component of the gut flora enhance systemic infection by aerobic or facultative anaerobic bacteria, and this facilitates mortality after irradiation.     

The nitrite reductase gene of Pseudomonas aeruginosa: Effect of growth conditions on the expression and construction of a mutant by gene disruption

Abstract The expression of nitrite reductase has been tested in a wild-type strain of Pseudomonas aeruginosa (Pao1) as a function of nitrate concentration under anaerobic and aerobic conditions. Very low levels of basal expression are shown under non-denitrifying conditions (i.e. absence of nitrate, in both aerobic and anaerobic conditions); anaerobiosis is not required for high levels of enzyme production in the presence of nitrate. A Pseudomonas aeruginosa strain, mutated in the nitrite reductase gene, has been obtained by gene replacement. This mutant, the first of this species described up to now, is unable to grow under anaerobic conditions in the presence of nitrate. The anaerobic growth can be restored by complementation with the wild-type gene.     

Experimental criteria for interpretation of bacterial sensitivity to dioxidine determined by diffusion from the disks

Antibacterial activity of dioxidine against aerobic and facultative anaerobic organisms under conditions of anaerobiosis i. e. conditions really observed for example in abscess cavities or necrotic tissues is 30 to 100 times as high as that under aerobic conditions. There is a relationship between sensitivity of bacteria to dioxidine under aerobic and anaerobic conditions which is expressed by the regression equation. Therefore, comparison of the MICs determined under anaerobic conditions with the growth inhibition zones formed by disks with the drug under aerobic conditions is possible. The MIC of dioxidine was determined under anaerobic conditions for 179 clinical strains of aerobic and facultative anaerobic bacteria and the growth inhibition zones of the same bacteria under aerobic conditions were evaluated with the use of disks containing 100, 75, 50, 25, 20, and 15 micrograms of the drug. The border line. MIC differentiating between resistant and sensitive strains was chosen to be equal to 4 micrograms/ml. Differentiation of the strains into sensitive and resistant ones by the values of the growth inhibition zones was performed with the method of error minimization described by C. Metzler and R. De Haan in 1974. Disks containing 25 micrograms of the drug allowed one to differentiate the strains under aerobic conditions into sensitive and resistant ones: the growth inhibition zones greater than 11 mm corresponded to the sensitive strains (the MIC smaller than 4 micrograms/ml) and the growth inhibition zones smaller than 11 mm corresponded to the resistant strains (the MIC greater than 4 micrograms/ml).     

Studies on Assembly of Vanadium-iron Protein in Vitro

By treating the reduced MoFe protein of nitrogenase from Azotobacter vinelandii with ophenanthroline under anaerobic or aerobic condition,inactive MoFe protein which was partialy deficient in both P-cluster and FeMoco could be obtained. After incubating the inactive MoFe protein with a reconstituent solution containing NaVO3,ferric homocitrate, Na2S and dithiothreitol, a reconstituted protein could be obtained. The proton reduction activity and absorption spectrum of the reconstituted protein could be well restored, but its C2H2-reduction activity could not be recovered. Its CD spectrum could be recovered except for the 550nm to 650nm region which differed from that of the reduced MoFe protein. The results showed that the reconstituted protein was different from MoFe protein,but was similar to vanadium-iron protein.     

Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis

Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.     

Bacteriological study of otogenic cerebral abscesses: chemotherapeutic role of metronidazole.

In nine consecutive patients with otogenic cerebral abscesses a mixed growth of aerobic and obligate anaerobic bacteria was isolated from the pus in five patients, and in the remaining four obligate anaerobes were the sole isolates. The commonest obligate anaerobe isolated was Bacteroides fragilis, which was present in all but one patient. The patients were all treated with metronidazole for the anaerobic organisms and with appropriate chemotherapy against the aerobic organisms isolated. All the patients recovered and only one was left with a neurological deficit. As otogenic cerebral abscesses constitute a major proportion of all cerebral abscesses, the use of metronidazole against obligate anaerobic bacteria, which tend to dominate in such abscesses, should reduce the high mortality from this condition.     

The role of anaerobic bacteria in chronic suppurative otitis media in children: Implications for medical therapy

This review describes the microbiology, diagnosis and medical management of chronic suppurative otitis media (CSOM) in children highlighting the role of anaerobic bacteria. In studies that employed adequate method for recovery of anaerobic bacteria polymicrobial aerobic and anaerobic flora was isolated from over half of the children with CSOM. The predominant aerobic isolates were Staphylococcus aureus and Pseudomonas aeruginosa and the most frequently isolated anaerobic organisms were Peptostreptococcus, Fusobacterium spp. and pigmented Prevotella and Porphyromonas spp. Several studies illustrated the efficacy of anti-infective agents effective against anaerobic bacteria in the treatment of CSOM. The medical therapy of CSOM should be directed at the eradication of the pathogenic aerobic and anaerobic organisms.     

The effect of oxygen on denitrification in Paracoccus denitrificans and Pseudomonas aeruginosa

Denitrification by Paracoccus denitrificans and Pseudomonas aeruginosa was studied using quadrupole membrane-inlet mass spectrometry to measure simultaneously and continuously dissolved gases. Evidence was provided for aerobic denitrification by both species: in the presence of O2, N2O production increased in Pa. denitrificans, while that of N2 decreased; with Ps. aeruginosa, the concentrations of both N2 and N2O increased on introducing O2 into the gas phase. Disappearance of NO-3 was monitored in anaerobically and aerobically grown cells which were maintained either anaerobically or aerobically: the rate and extent of NO-3 utilization by both species depended on growth and maintenance conditions. The initial rate of disappearance was most rapid under completely anaerobic conditions, and lowest rates occurred when cells were grown anaerobically and maintained aerobically. In nitrogen balance experiments both species converted over 87% of the added NO-3 to N2 and N2O under both anaerobic and aerobic maintenance conditions.