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Isolation and identification of intestinal steroid-desulfating bacteria from rats and humans.

We isolated 12 strictly anaerobic steroid-3-sulfate-desulfating strains from the intestinal floras of rats and humans. Two strains (S1 and S2) of the same atypical Clostridium species and an atypical Lactobacillus strain (termed R9) were obtained from rats. The human isolates were identified as Eubacterium cylindroides (two strains, H1 and H2), Peptococcus niger (two strains, H4 and H89), and Clostridium clostridiiforme. We also isolated, from different human fecal samples, four strains of phenotypically similar asaccharolytic Bacteroides strains, H6.2a, H6.2b, H65, and H175. Aryl steroid sulfatase activity for estrogen sulfates was present in all isolates. Alkyl steroid sulfatase activity for both 3 alpha- and 3 beta-sulfates was found only in P. niger H4. The same P. niger strain and Clostridium strains S1 and S2 also possessed bile acid sulfatase activity.

Animals↗

Phosphatase activity of anaerobic organisms.

Anaerobic organisms were tested for phosphatase activity in different pH ranges. Several groups of organisms displayed characteristic patterns. Bacteroides fragilis, B. melaninogenicus, and B. ruminicola produced phosphatase with strongest activity at pH 8.6. Fusobacterium mortiferum was the only species of this genus to show strong hydrolysis. The enzyme was active in both acid and alkaline ranges. The activity of gram-positive organisms was variable, the most active groups being Clostridium perfringens, Peptostreptococcus intermedius, P. micros, and Peptococcus constellatus. The incorporation of phosphatase activity into the identification scheme of anaerobes seems feasible. There was a correlation of hydrolysis with several important pathogens.

Anaerobiosis↗

Differential effects of oxygen and oxidation-reduction potential on the multiplication of three species of anaerobic intestinal bacteria.

The sensitivity of three strains of anaerobic intestinal bacteria, Clostridium perfringens, Bacteroides fragilis, and Peptococcus magnus, to the differential effects of oxygen and adverse oxidation-reduction potential was measured. The multiplication of the three organisms was inhibited in the presence of oxygen whether the medium was at a negative oxidation-reduction potential (Eh of -50 mV), poised by the intermittent addition of dithiothreitol, or at a positive oxidation-reduction potential (Eh of near +500 mV). However, when these organisms were cultured in the presence of oxygen, no inhibition was observed, even when the oxidation-reduction potential was maintained at an average Eh of +325 mV by the addition of potassium ferricyanide. When the cultures were aerated, the growth patterns of the three organisms demonstrated different sensitivities to oxygen. P. magnus was found to be the most sensitive. After 2 h of aerobic incubation, no viable organisms could be detected. B. fragilis was intermediately sensitive to oxygen with no viable organisms detected after 5 h of aerobic incubation. C. perfringens was the least sensitive. Under conditions of aerobic incubation, viable organisms survived for 10 h. During the experiments with Clostridium, no spores were observed by spore staining.

Anaerobiosis↗

Animal model for anaerobic lung abscess.

There are no satisfactory animal models for the study of anaerobic lung abscess. Aspiration of food, gastric mucin, or hydrochloric acid, or any combination of these, along with oropharyngeal bacteria, is commonly believed to cause aspiration pneumonia and lung abscess. In the animal model described, none of the adjuvants was effective in producing anaerobic lung abscesses. Anaerobic bacteria derived from dental scrapings of a healthy adult (Peptococcus morbillorum, Fusobacterium nucleatum, Eubacterium lentum, and Bacteroides fragilis), when inoculated transtracheally without any adjuvants into New Zealand male white rabbits, consistently produced lung abscesses. Neither B fragilis by itself nor a mixture of P. morbillorum, F. nucleatum, and E. lentum without the addition of B. fragilis produced lung abscesses. The bacterial isolates used in this study were stored in prereduced chopped-meat-glucose medium and subcultured several times and were found effective in reproducing anaerobic lung abscesses repeatedly. This animal model is suitable for the study of pathogenesis, diagnosis, and treatment of B. fragilis-associated anaerobic lung abscess.

Aerosols↗

Pathogenicity of anaerobic gram-positive cocci.

The pathogenicity of 20 strains of facultative or anaerobic gram-positive cocci (AGPC) was investigated by injecting them alone or mixed with other flora into mice, utilizing the subcutaneous abscess model. Abscesses induced by a mixture of two organisms were uniformly larger than those induced by single organisms. The relationships among seven AGPC strains, eight aerobes, and two Bacteroides spp. were determined by treating the infected animals with antibiotics and observing the effect of therapy directed against one or both organisms present in the abscess. A total of 70 different combinations were tested. As judged by their responses to antimicrobial therapy, facultative cocci or AGPC were relatively more important than the other species in 6 combinations, equally important in 35 combinations, and less important in 29 combinations. The AGPC most often found to be equal to or more important than the other bacteria were Peptococcus magnus, Streptococcus constellatus, and Peptostreptococcus anaerobius. Proteus mirabilis, Escherichia coli, and Staphylococcus aureus were frequently found to be of more importance than the AGPC.

Abscess↗

Bactericidal activity of aerobic and anaerobic polymorphonuclear neutrophils.

Human polymorphonuclear neutrophils (PMN) were made anaerobic by nitrogen washout (oxygen saturation <1%, Eh < -42 mV at pH 7.0), and the ability of the cells to kill bacteria was assayed and compared to the bactericidal activity of aerobic PMN. Anaerobic PMN were able to kill Staphylococcus epidermidis, Enterococcus, viridans streptococci, Pseudomonas aeruginosa, Peptostreptococcus anaerobius, Bacteroides fragilis, Clostridium perfringens, and Peptococcus magnus normally. Organisms that were not killed normally by anaerobic PMN included Staphylococcus aureus (strains Wood 46 and 502a), Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Proteus vulgaris, and Salmonella typhimurium. These findings suggest that mechanisms other than those dependent on hydrogen peroxide may be important in the killing of some bacteria.

Aerobiosis↗

Two pathways of glutamate fermentation by anaerobic bacteria.

Two pathways are involved in the fermentation of glutamate to acetate, butyrate, carbon dioxide, and ammonia-the methylaspartate and the hydroxyglutarate pathways which are used by Clostridium tetanomorphum and Peptococcus aerogenes, respectively. Although these pathways give rise to the same products, they are easily distinguished by different labeling patterns of the butyrate when [4-(14)C]glutamate is used as substrate. Schmidt degradation of the radioactive butyrate from C. tetanomorphum yielded equally labeled propionate and carbon dioxide, whereas nearly all the radioactivity of the butyrate from P. aerogenes was recovered in the corresponding propionate. This procedure was used as a test for the pathway of glutamate fermentation by 15 strains (9 species) of anaerobic bacteria. The labeling patterns of the butyrate indicate that glutamate is fermented via the methylaspartate pathway by C. tetani, C. cochlearium, and C. saccarobutyricum, and via the hydroxyglutarate pathway by Acidaminococcus fermentans, C. microsporum, Fusobacterium nucleatum, and F. fusiformis. Enzymes specific for each pathway were assayed in crude extracts of the above organisms. 3-Methylaspartase was found only in clostridia which use the methylaspartate pathway, including Clostridium SB4 and C. sticklandii, which probably degrade glutamate to acetate and carbon dioxide by using a second amino acid as hydrogen acceptor. High levels of 2-hydroxyglutarate dehydrogenase were found exclusively in organisms that use the hydroxyglutarate pathway. The data indicate that only two pathways are involved in the fermentation of glutamate by the bacteria analyzed. The methylaspartate pathway appears to be used only by species of Clostridium, whereas the hydroxyglutarate pathway is used by representatives of several genera.

Acetates↗

Effect of bacterial contamination on cecal size and cecal contents of gnotobiotic rodents.

In the present investigation the effect of various bacterial contaminations of gnotobiotic mice and rats on cecal size is presented. Of the species tested, Bacteroides oralis and Fusobacterium nucleatum did not establish in germ-free mice. Streptococcus mutans, Clostridium difficile, a Neisseria strain and two recent cecal isolates established, but failed to exert an effect upon the cecum of mice. A group K streptococcus and B. fragilis increased the cecal size apparently by increasing the levels of water-soluble protein, peptides, and carbohydrates in the cecal contents. Mixed ileal bacteria decreased the cecal size by preventing accumulation of soluble proteins and carbohydrates in the cecum. A Peptococcus strain caused a reduction by lowering the levels of insoluble material in the cecum. When this strain was combined with two Clostridium isolates and introduced into gnotobiotic rats, 50 to 65% cecal reduction was observed. This polycontamination did not decrease the per cent water of the cecal contents but caused lower levels of both soluble and insoluble material to accumulate in the cecum. No net nitrogen absorption from the distal small intestine occurred in either the germ-free or polycontaminated rats.

Animals↗

Production of deoxyribonuclease, ribonuclease, coagulase, and hemolysins by anaerobic gram-positive cocci.

Clinical isolates of Peptococcus and Peptostreptococcus species and Streptococcus intermedius strains were obtained from local hospitals. After confirmed identification, each isolate was tested for the in vitro production of deoxyribonuclease, ribonuclease, coagulase, and hemolysins. Of the 60 strains studied, 18 had enzymatic activity. The variability of enzyme production suggests that such assays are not suitable as an aid to identification of these organisms.

Anaerobiosis↗

Achromopeptidase for lysis of anaerobic gram-positive cocci.

Achromopeptidase, which has potent bacteriolytic activity for most of the gram-positive aerobic bacteria, was for the first time used for the lysis of anaerobic cocci. Most of the lysozyme-resistant gram-positive anaerobic cocci were lysed with this new enzyme. Peptococcus magnus was the only organism tested resistant to achromopeptidase. P. saccharolyticus was quite unusual because it was very sensitive to both achromopeptidase and lysozyme.

Anaerobiosis↗

Efficacy of direct Gram stain in differentiating staphylococci from streptococci in blood cultures positive for gram-positive cocci.

A preponderance of clusters seen on direct Gram stain of blood cultures positive for gram-positive cocci was 98% sensitive and 100% specific for identification of staphylococcal species or of Peptococcus. A preponderance of chains, pairs, or both was 100% sensitive and 98% specific for identifying streptococci. Further presumptive identification of either staphylococci or streptococci based on microscopic morphology was unreliable. The direct Gram stain is highly reliable for differentiating staphylococci from streptococci and should be of considerable value to clinicians selecting initial antimicrobial therapy.

Blood↗

Comparative efficacy of four antibiotics in anaerobic pulmonary infection. An experimental model in rabbits.

The efficacy of cefoxitin, mezlocillin, latamoxef and metronidazole in anaerobic lung infection was studied using a rabbit model. A mixture of Bacteroides fragilis, Peptococcus morbillorum, Eubacterium lentum and Fusobacterium nucleatum was inoculated transtracheally to produce infection within the lung. Mezlocillin was most effective, achieving bacteriologic cure in 5 out of 8 animals. With cefoxitin therapy, 4 out of 8 became bacteriologically sterile. Severe diarrhea with elevated titers of Clostridium difficile toxin was noted in most cefoxitin-treated animals. Latamoxef- and metronidazole-treated animals had apparently healed lesions, but cultures were positive in 6 and 7 out of 8 in each group, respectively. The commonest pathogen isolated in the last two groups was P. morbillorum. The therapeutic superiority of mezlocillin over metronidazole and latamoxef was statistically significant (p less than or equal to 0.05).

Animals↗

Organic acids and anaerobic microorganisms in the contents of the cholesteatoma sac.

Organic acids in the contents of the cholesteatoma sac from 28 cases were studied by gas chromatographic technique. Five volatile fatty acids (acetate, propionate, isobutyrate, butyrate and isovalerate) and lactate were detected in large amounts, which may lower the pH of the cholesteatoma content. These acids were considered to be derived from products of anaerobic microorganisms. Therefore, the contents from 12 cases were cultured anaerobically in a glove box. Obligate microorganisms were identified in 92% of the cases and Peptococcus, Bacteroides, and Clostridium species were frequently isolated. In vitro, such obligate anaerobes produced various organic acids from the cholesteatoma content. Facultatives such as Staphylococcus aureus and Proteus mirabilis produced acetate in the content under aerobic and anaerobic conditions, whereas no organic acid was produced by Pseudomonas aeruginosa. Organic acids in the cholesteatoma content could be fermentative products made by the microorganisms, anaerobes and facultatives, which use the content as a substrate for acid production.

Acetates↗

Studies of anaerobic bacteria in chronic otitis media.

In the 760 cases of chronic otitis media studied from 1970 to 1976, the detection ratio of anaerobic bacteria and the relation between the nature of anaerobic bacteria and the conditions of focus were investigated. In 8.2% of 760 cases 9 kinds and 81 strains of anaerobic bacteria was detected. Most of the anaerobic bacteria were Peptococcus sp. or Bacteroides sp. and in general non-sporogenic anaerobic bacteria accounted for 97.5%. Anaerobes were frequently detected in cases where the middle ear cavity was filled with cholesteatoma or granulation and in cases of infection recurring after operation.

Bacteroides Infections↗

Bacteriological study in chronic maxillary sinusitis.

The bacterial findings of 73 maxillary sinuses in 48 patients with chronic maxillary sinusitis, together with 7 non-inflamed sinuses, are reported. Employing an intraoperative technique and simultaneous collection of 3 different types of specimens in the present investigation made possible comparison of their bacterial characteristics. The results indicated that intraoperative culture of antral mucosa seems to provide the most reliable finding of bacterial flora in chronic maxillary sinusitis. The anaerobic bacteria were never found in the mucosal culture of non-inflamed sinus. The anaerobes appeared to invade the sinus cavity following the sealing of the ostium through the lymphatic or venous system and maintain the inflammatory process. Microbiological analysis of the results between the infected and non-inflamed sinuses established anaerobic bacteria as the most important pathogen in chronic maxillary sinusitis. The predominant anaerobes recovered in descending order of frequency were Veillonella sp., Peptococcus sp., Propionibacterium acne and anaerobic nonspore-forming GPB. Statistical analysis of the results of mucosal culture of inflamed and control materials demonstrated that those aerobic and faculatative bacteria recovered in the inflamed sinus appeared to be the normal inhabitants of non-inflamed sinus mucosa. The presence of normal flora in the normal healthy sinus mucosa may explain the chain of events that follows the occlusion of the ostium. These aerobic bacteria may become pathogenic and play a role in the pathogenesis of sinusitis.

Adolescent↗

The microbial flora in venous leg ulcers without clinical signs of infection. Repeated culture using a validated standardised microbiological technique.

The change of ulcer size in relation to the presence of species and quantities of microorganisms was analysed in 58 patients with venous leg ulcers, all without clinical signs of infection. Microbiological samples were taken on the day of inclusion and then repeated 4 times at monthly intervals or until the ulcer had healed or was too small to be cultured from. There was growth of microorganisms in all ulcers, and the numbers were below 10(4) per mm2 of ulcer surface in all cases. No correlation was found between ulcer size change and the species and amounts of microorganisms. Sixty-nine species were isolated. Staphylococcus aureus was found in 88%, Enterococcus faecalis in 74%, Enterobacter cloacae and Peptococcus magnus in 29%, and fungi in 11% of the samples. One or more obligate anaerobe species was found in 41% of the samples and in half of the ulcers and constituted 62% of all bacterial species. The colonising ulcer flora was markedly constant over time in the individual ulcers regardless of change in size. Resident bacterial species were found in 57 of the 58 ulcers. If all samples were considered, the microorganisms were associated with not more than one fifth of the variability in healing rate, as shown by linear multiple regression analysis. The same species of microorganisms were found in ulcers that decreased (or healed) and in those that increased in size. Although an association between the microorganisms and ulcer healing could not be ruled out in this study, there seems to be no indication for routinely performed culture in the absence of clinical signs of infection in venous leg ulcers.

Aged↗

Microbiological studies of the bacterial flora of the external auditory canal in children.

The aerobic and anaerobic bacterial flora of the external auditory canal (EAC) was studied in 72 children. Aerobes only were isolated in 58 patients (80%) and anaerobic bacteria only in 2 (3%). Mixed aerobic and anaerobic isolates were recovered in 12 cases (17%). There were 122 aerobic and 15 anaerobic isolates accounting for 1.7 aerobes and 0.2 anaerobes per specimen. The most common aerobic isolates were Staphylococcus epidermidis (56 isolates), alpha hemolytic streptococcus (13) and Pseudomonas aeruginosa (8). The two anaerobic organisms recovered were Proprionibacterium acnes (13 isolates) and Peptococcus sp. (2). These findings demonstrate the polymicrobial bacterial flora of EAC in children where aerobic facultative and anaerobic bacteria are part of the normal flora.

Bacteria↗

Recurrent anaerobic skin abscesses.

20 adult patients (17 women) presented axillary, gluteal, external genital, breast and groin abscesses. Anaerobic bacteria were isolated in 10 cases; Peptococcus magnus in 8. staphylococcus aureus was the sole organism in 2 cases, skin flora (Staphylococcus epidermidis) in 6. No bacteria were isolated from 2 patients. The Staph. aureus were mainly phage group III strains or nontypable. It is apparent that these kinds of skin infections comprise a special group with regard to epidemiology and etiology.

Abscess↗