Antibiotic susceptibility patterns as aids in classification and characterization of gram-negative anaerobic bacilli

Patterns of susceptibility of gram-negative anaerobic bacilli to antibiotics have been found to be distinctive and of significant help in classification and identification. Five major groups of gram-negative anaerobic bacilli have been defined on the basis of morphological and biochemical criteria. Antibiotic sensitivity patterns conform to these groupings and provide additional taxonomic criteria. The Bacteroides fragilis group is resistant to penicillin G, whereas the other groups are generally sensitive. B. fragilis strains are relatively sensitive to erythromycin, whereas the Sphaerophorus necrophorus group is resistant. B. melaninogenicus strains, B. oralis, and Fusobacterium are all more sensitive to kanamycin and neomycin than the other two groups. Kanamycin is more active against Fusobacterium strains than neomycin, but less active against all other groups. Colistin or polymyxin B is useful for distinguishing between the resistant B. fragilis and the sensitive S. necrophorus. Antibiotic susceptibility determinations may be more readily performed in clinical laboratories than certain biochemical tests recommended for differentiation of the gram-negative anaerobic bacilli and may serve as helpful adjuncts to morphological and biochemical observations in classifying and characterizing these organisms. The use of standardized procedures for antibiotic susceptibility tests is essential if comparable results are to be obtained in different laboratories.     

The Characterization of Clinically Important Gram Negative Anaerobic Bacilli by Conventional Bacteriological Tests

One hundred and sixty-five reference strains and laboratory isolates of Gram negative, non-sporing, anaerobic bacilli were subjected to a series of simple laboratory tests that were initially selected for their discriminatory value. Conventional biochemical tests, tests of resistance to antibiotics, and tolerance to dyes and bile salts were included. These tests allowed a clear separation of strains into three main groups: Bacteroides fragilis, B. melaninogenicus and Fusobacterium spp. Certain tests were found useful for identifying recognized subspecies of B. fragilis and B. melaninogenicus . A scheme for the identification of unknown laboratory isolates of Gram negative anaerobic bacilli is presented.     

Growth of Bacteroidaceae in Stirred Fermentors

The conditions for increasing bacterial yields in cultures of Bacteroidaceae by the use of stirred fermentors and pH control were investigated by means of three representative species: Sphaerophorus necrophorus, Bacteroides fragilis, and B. melaninogenicus. A medium containing tryptone, yeast extract, and glucose or sucrose was used. Horse serum had to be added to obtain substantial growth of B. melaninogenicus. The optimal pH for growth rate and yield was 7.0 to 7.2. Lysis of the bacteria occurred when the glucose (or sucrose) was exhausted. The rate of lysis was very high in cultures of S. necrophorus, less so in B. fragilis and B. melaninogenicus. Pleomorphism, manifested as large sperical forms of the bacteria, was observed in the late logarithmic phase of S. necrophorus. Great differences in the length of the lag phase and of the mean generation time were found among the three bacterial species. The yield in static cultures of the three species without pH control was approximately 0.4 g of dry cells per liter, but was increased, in stirred fermentors with pH control, to 3.5 g (S. necrophorus), 2.7 g (B. fragilis), and 4.3 g (B. melaninogenicus) per liter. With an inoculum density of 5 to 10 mg (dry weight) per liter, these yields were obtained in approximately 10 (S. necrophorus), 25 (B. fragilis), and 35 hr (B. melaninogenicus), respectively.     

Sphaerophorus Necrophorus a Study of 23 Strains

Twenty-three strains of Sphaerophorus necrophorus, isolated from animal pathological processes, were characterized. The two biotypes of Fievez (1963) were recognized, differing in colony morphology, cell morphology, degree of hemolysis, growth modus in broth, and hemagglutination. The following characters were shared by all strains: Gram-negative, anaerobic, nonsporeforming, immotile rods, producing acid from fructose, glucose, mannose, maltose, and (variably) from galactose, but not from arabinose, xylose, lactose, saccharose, cellobiose, melezitose, adonitole, sorbitole, mannitole, and salicine. Terminal pH in glucose medium was 5.77 ± 0.17. Growth was not inhibited by 0.008 % brilliant green, but was partially inhibited by 10 % ox bile. Indole was produced. Nitrate was not reduced. A lecitinase was present. Proteolytic properties were present. Threonine was deaminated. The fatty acids: acetic, propionic, and butyric acid were produced in medium both with and without glucose.     

Gram-Negative Anaerobes in the Intestinal Flora of Pigs

A differential count of the gram-negative, anaerobic, non sporeforming bacteria in different segments of the pig intestinal tract was performed in 2 groups of pigs. Seventy-seven strains were identified, belonging to 6 groups: Sphaerophorus necrophorus, anhemolytic Sphaerophorus spp., Bacteroides (Eggerthella) spp., Fusobactenum spp., Veillonella/Acidaminococcus spp., and Peptostreptococcus elsdenii. The characters of these groups are described, and quantitative data on their occurrence in a group of normal porkers and a group of pigs with experimental swine dysentery are given.     

The Hydrolysis of Dextran by Gram Negative Non-sporing Anaerobic Bacilli

The ability of Gram negative anaerobic bacilli to hydrolyse dextran was determined in liquid and solid media containing Blue Dextran 2000. Released blue chromophore in the liquid medium was detected spectrophotometrically. Results obtained with 334 strains of Bacteroidaceae grown on the solid medium indicated that most strains did not hydrolyse the substrate. Hydrolysis of Blue Dextran 2000 occurred with certain strains of Bacteroides thetaiotaomicron , B. melaninogenicus ss. melaninogenicus, B. oralis, B. ovatus and B. ochraceus.     

Effect of selective media on loss of congo red binding inShigella flexneri

Summary The effect of growth ofShigella flexneri on various selective media on retention of congo red (CR) binding ability was determined to evaluate the effectiveness of isolation techniques regarding maintenance of the virulence plasmid. WhenS. flexneri was surface-plated onto selective agars and the resulting colonies replica plated onto CR plates, no white colonies indicative of loss of virulence were found despite repeated trials. However, whenS. flexneri was grown in liquid media (agar was removed from agar-containing media by centrifugation), white colonies were found upon plating onto CR plates. Most common selective media for shigellae produced fewer than 5–10 white colonies/1000 red colonies. However, growth in broth prepared from violet red bile agar, desoxycholate citrate agar, and SS agar gave more than 100 white colonies/1000 red colonies. Loss of CR binding was demonstrated whenS. flexneri was grown in broth containing tergitol 7, sodium dodecyl sulfate, bile salts #3, crystal violet, eosin y or methylen blue. However, concentrations of selective agents that led to loss of CR binding were much higher than those used in selective media. Results indicate that under usual conditions of isolation ofS. flexneri from food and clinical specimens, CR binding appears to be a relatively stable character with most selective media; however, use of violet red bile agar, desoxycholate citrate agar, and SS agar may lead to substantial loss of congo red binding indicating that the isolates may not be virulent.     

Comparison of Schaedler Agar and Trypticase Soy-Yeast Extract Agar for the Cultivation of Anaerobic Bacteria

Schaedler agar (SA) and Trypticase soy-yeast extract agar (TSYEA), both supplemented with rabbit blood (5%, v/v) and menadione (0.5 mg/liter), were compared with respect to quantitative recovery, quality of growth, and rapidity of growth of selected anaerobic bacteria. The media were stored for 2 to 4 days prior to use in an anaerobic glove box, where all subsequent bacteriological procedures were performed. After 24 hr of incubation, colonies of Clostridium cadaveris (C. capitovale), C. haemolyticum, C. novyi A, and C. perfringens were larger on SA than on TSYEA, and the appearance of C. novyi B colonies on SA at 24 hr antedated their appearance on TSYEA. Quantitative recovery of C. novyi B was improved on SA; recovery of the other clostridia tested was comparable on the two media (inconclusive results were obtained with C. novyi A). Rough colonial types of some of the clostridia emerged on SA. No appreciable differences in results with the two media were noted for Bacteroides fragilis, B. melaninogenicus, or Fusobacterium fusiforme.     

Dehydrogenase Patterns in the Study of Bacteroidaceae

Enzyme patterns were obtained by starch-gel electrophoresis of cell-free extracts from organisms in the family Bacteroidaceae. Esterases, phosphatases, and lactic and succinic dehydrogenases were detected but offered no possibility for classification purposes. Alpha-glycerophosphate dehydrogenase and 6-phosphogluconate dehydrogenase were not detected. Zymograms of malic, glutamic, and glucose-6-phosphate dehydrogenases separated Bacteroides species from Sphaerophorus and Fusobacterium species. Malic dehydrogenase and glucose-6-phosphate dehydrogenase were found in Bacteroides but not Sphaerophorus and Fusobacterium. These dehydrogenase zymograms placed three gram-negative, nonsporeforming anaerobic rod isolates with the Bacteroides species. A close correlation was found between the classification of Bacteroidaceae by zymogram analysis and a numerical taxonomy scheme previously published from this laboratory.     

Anaerobic gram-negative faecal flora in patients with Crohn's Disease and healthy subjects

The anaerobic gram-negative faecal flora of five patients with Crohn's Disease (CD) was identified and compared with that of healthy subjects. For isolation and cultivation of the anaerobic gram-negative rods a non-selective medium was used. There were no significant differences in numbers of Bacteroides and Fusobacterium spp. between patients with CD and healthy subjects. However, the numbers of the "Bacteroides fragilis" group were significantly higher in patients than in controls. The high numbers of the "B. fragilis" group in the faeces of patients were particularly due to B. vulgatus which was 6 times more frequent in patients than in healthy subjects. This indicated that B. vulgatus was responsible for the higher numbers of anaerobic gram-negative rods in the faecal flora of patients with CD.     

Tryptic Soy Bile-Kanamycin Test for the Identification of Bacteroides fragilis

A simple and practical test for the identification of Bacteroides fragilis is described. It utilizes two well-known properties of this species, i.e., stimulation of growth by bile and resistance to kanamycin. The test media are a tryptic-soy bile agar plate and a supplemented blood agar plate on which a kanamycin 1,000-mug/ml disk is placed. Incubation is for 24 h at 37 C in GasPak. The results of screening 190 strains, mostly clinical isolates, indicate that B. fragilis can be easily and reliably distinguished from other Bacteroides and from Fusobacterium species by its growth on tryptic-soy bile agar and resistance to kanamycin.     

Oxygen Sensitivity of Various Anaerobic Bacteria

Anaerobes differ in their sensitivity to oxygen, as two patterns were recognizable in the organisms included in this study. Strict anaerobes were species incapable of agar surface growth at pO(2) levels greater than 0.5%. Species that were found to be strict anaerobes were Treponema macrodentium, Treponema denticola, Treponema oralis n. sp., Clostridium haemolyticum, Selenomonas ruminatium, Butyrivibrio fibrisolvens, Succinivibrio dextrinosolvens, and Lachnospira multiparus. Moderate anaerobes would include those species capable of growth in the presence of oxygen levels as high as 2 to 8%. The moderate anaerobes could be exposed to room atmosphere for 60 to 90 min without appreciable loss of viability. Species considered as moderate anaerobes were Bacteroides fragilis, B. melaninogenicus, B. oralis, Fusobacteria nucleatum, Clostridium novyi type A, and Peptostreptococcus elsdenii. The recognition of at least two general types of anaerobes would seem to have practical import in regard to the primary isolation of anaerobes from source material.     

Empirical antibiotic therapy of wounds complicated by anaerobic non-clostridial infection

Antibacterial activity of 14 drugs against clinical strains of asporogenic anaerobes causing wound infections in the soft tissues i. e. Bacteroides fragilis and Bacteroides melaninogenicus as well as anaerobic gram-positive++ cocci was assayed with the method of serial dilutions in agar. It was shown that among the investigated species B. fragilis had the most marked resistance since out of the 14 drugs only 8 were sufficiently active against it i.e. carbenicillin, levomycetin, lincomycin, dioxidine, metronidazole, thinidazole, nitrazole and erythromycin. The choice of drugs for treating infections caused by B. melaninogenicus and anaerobic grampositive cocci unlike those caused by B. fragilis offered no difficulty since practically++ all the investigated drugs were highly active against the causative agents. There was observed relationship between the frequency of asporogenic anaerobes and the wound genesis. The characteristic features of the species composition connected with localization of the suppurative foci were indicated. The detected specific antimicrobial profiles of the asporogenic anaerobes causing wound infections and the peculiarity of their participation in development of purulent infections of the soft tissues provided a differential approach to empirical antibacterial therapy prior to the pathogen bacteriological investigation and availability of the antibioticograms.     

Application of Immunofluorescence to Studies on the Ecology of Sphaerophorus necrophorus

Sphaerophorus necrophorus (bovine liver abscess isolates) antiserum was fractionated and labeled with fluorescein isothiocyanate. The fluorescent-antibody (FA) conjugate proved to be species-specific and facilitated the detection of S. necrophorus cells in liver abscesses, viscera, and ruminal contents of cattle. Brightly fluorescing S. necrophorus cells were observed in experimentally inoculated soil incubated anaerobically at 37 and 4 C for 8 and 10 months, respectively. When incubated under moist conditions (80% water holding capacity) at 37 C, the test organism survived in both sterile and unsterile soil for as long as 8 weeks. Results strongly support the feasibility of using FA techniques concurrent with cultural methods for rapid detection of S. necrophorus infections.     

Extracellular Deoxyribonuclease Production by Anaerobic Bacteria

The production of extracellular deoxyribonuclease was examined with anaerobic organisms isolated from clinical specimens. Nuclease activity was extraordinarily common. All strains of Fusobacterium, including eight species, as well as Bacteroides fragilis and B. melaninogenicus, displayed enzyme activity. Whereas the gram-positive bacteria were generally less productive, all strains of Clostridium perfringens, Peptostreptococcus intermedius, and P. anaerobius specifically produced deoxyribonuclease. The test is taxonomically valuable, particularly in the characterization of gram-positive cocci, since a deoxyribonuclease-producing coccus indicates P. intermedius or P. anaerobius. Additionally, possession of the enzyme may prove to be a useful correlate of the potential pathogenicity of anaerobes.     

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.     

The Classification of Bacteroides melaninogenicus and Related Species

One hundred and seventy-five strains of Bacteroides melaninogenicus , 17 strains of B. oralis and six strains of B. ochraceus were studied in a series of biochemical, chemical tolerance and antibiotic disc resistance tests and by the gas-liquid chromatographic analysis of the acid end products of metabolism. Strains of B. melaninogenicus ss. asaccharolyticus formed a distinct group with clear differences from other B. melaninogenicus strains. B. melaninogenicus ss. intermedius strains formed a homogeneous group that could be readily identified. B. ochraceus was distinguished from other Bacteroides spp. by its ability to grow in air enriched with CO₂. Bacteriodes melaninogenicus ss. melaninogenicus and B. oralis gave very similar patterns of results with the tests used and invariably were indistinguishable; the capacity to produce black-pigmented colonies on blood-containing media may not be a valid criterion for dividing these similar strains into two species.     

慢性根尖周炎的细菌学研究

本文对14例慢性根尖周炎患者16个感染根管细菌培养检查的结果发现:以厌氧菌为优势的混合菌感染是慢性根尖周炎感染根管细菌学的主要特点。穿髓根管由于敞开的髓腔,同一根管内4—9种细菌混合感染者占87.5％;未穿髓的感染根管2—3种细菌混合感染的占62.5％,4种以上细菌混合感染的占37.5％。优势的感染菌包括产黑色素类杆菌、口腔类杆菌、口类杆菌、具核梭杆菌、干酪乳杆菌、内氏放线菌等6种厌氧菌;血液链球菌、变链球菌和酿脓链球菌为最常见的兼性厌氧菌。本文指出口腔正常菌群是感染根管的机会病原菌。     

Deoxyribonucleic acid base composition of certain species of the genus Bacteroides

The moles percent guanine plus cytosine content of the DNA (% G + C) of 15 Bacteroides strains representing six species was determined. One group, including three strains of Bacteroides ruminicola, two strains of B. melaninogenicus, two strains of B. succinogenes, and one strain of B. oralis (JI), had a % G + C of 47.6--50.3 and a second group including two strains of B. amylophilus, four strains of B. fragilis, and one strain of B. succinogenes had a lower % G + C of 40.3--42.7. The taxonomic relationships among these bacteroides species were discussed.     

Isolation of Shigellae: VIII. Comparison of Xylose Lysine Deoxycholate Agar, Hektoen Enteric Agar, Salmonella-Shigella Agar, and Eosin Methylene Blue Agar with Stool Specimens

Two enrichment broths and four plating media were compared for efficiency of detection of enteric pathogens from 1,597 stool specimens. Of 170 salmonellae isolated from the composite of all methods, direct streaking yielded but 54%, whereas enrichment in gram-negative broth found 87% and Selenite-F broth 97%. By contrast, gram-negative broth produced 100% of the 17 shigellae, Selenite-F broth but 77%, and direct streaking only 59%. Thus, enrichment methods produced almost twice the number of both pathogens as direct streaking. Comparison of the plating media revealed xylose lysine deoxycholate agar (XLD) and Hektoen enteric agar to be equal in their abilities to find both pathogens. Both were moderately better than Salmonella-Shigella agar and markedly superior to eosin methylene blue agar. XLD fround 83% of salmonellae produced by the composite of four media and 90% of the shigellae. Hektoen enteric agar found 80% of both. Salmonella-Shigella agar detected 74 and 68%, respectively, and eosin methylene blue agar only 42 and 63%. The numbers of false positives accruing to each medium, however, showed Hektoen enteric and Salmonella-Shigella agars to produce more than twice as many false-positive plates as XLD. Similarly, Selenite-F broth resulted in many more false-positives for all plating media than did gram-negative broth. Consequently, the index of validity, which equates successful isolation of pathogens with total pickings, favored XLD and gram-negative broth as the media of choice, with direct streaking the poorest method by all counts.