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Comparison of three methods of susceptibility testing of bacteroidaceae, peptococcaceae and other anaerobes to doxycycline.

The in vitro activity of doxycycline against 147 strains of gram-positive and gram-negative anaerobes was determined by broth dilution, agar dilution and agar diffusion tests. The strains were isolated from clinical specimens in 1977. Saccharolytic Bacteroides strains (39 B. fragilis, 6 B. thetaiotaomicron, 4 B. vulgatus) showed broth dilution MICs of less than or equal to 0.0625-4.0 microgram/ml after 5 h incubation and MICs of less than or equal to 16 microgram/ml after 15 h incubation at 37 degrees C. With strains of B. oralis, B. melaninogenicus ss. intermedius, B. corrodens, Veillonella sp. and Megasphaera elsdenii the broth dilution MICs were less than or equal to 0.0625-32 microgram/ml after 15 h incubation. Peptostreptococcus spp., Propionibacterium acnes, Eubacterium sp., Bifidobacterium sp. and Clostridium sp. had broth dilution MICs of less than or equal to 2 microgram/ml, whereas Peptococcus spp. were inhibited by less than or equal to 0.0625 to greater than 32 microgram/ml. With the great majority of strains tested, MICs were 2 to 256 times higher in agar than in broth dilution tests. Clinical and experimental studies seem to be needed to determine which of the in vitro data are correlated to the outcome of doxycycline therapy. Several groups and species were separately considered for statistical analysis of the relationship between zone size and MIC. With gram-positive anaerobes, correlation was poor between broth dilution MIC and zone size (correlation coefficients r = - 0.168 for Peptococcaceae, and r = - 0.108 for P. acnes). When calculating the regression lines for agar dilution MICs and zone diameters, a higher correlation was found (r = - 0.9 for Peptococcaceae; r = - 0.397 for P. acnes). With B. fragilis and other Bacteroides species correlation coefficients were r = - 0.807 to r = - 0.891 for broth and agar dilution MICs and zone size but stochastic linearity was lacking.

Anaerobiosis↗

The taxonomy of the Peptococcaceae.

Current problems in the classification and identification of Peptococcaceae with special reference to clinically significant species are discussed briefly. Further research is needed to clarify most of the existing discrepancies.

Peptococcaceae↗

Peptococcaceae bacteremia.

Bacteremia with Peptococcaceae is an uncommon clinical manifestation of infection with this family of microorganisms. A 20-month review of 12 patients with bacteremic infections due to anaerobic gram-positive cocci revealed that obstetrical patients during the peripartum period constitute the group at greatest risk for the development of such infections. Eight of the 12 patients were young women hospitalized on the obstetrical service. Seven patients had postpartum endometritis and one patient had chorioamnionitis. The remaining four patients include a single patient each with pylephlebitis and microabscesses of the liver, ascending cholangitis, thoracic empyema, and necrotizing soft-tissue infection. Bacteriologic identification of the microorganisms revealed the following: Peptostreptococcus micros (5 patients), Peptostreptococcus asaccharolyticus (5 patients), Peptostreptococcus magnus (1 patient), and Peptococcus species (1 patient). Eleven of the 12 patients received appropriate antibiotic therapy. All patients did well and there were no major sequelae.

Adolescent↗

16S ribosomal DNA sequences of anaerobic cocci and proposal of Ruminococcus hansenii comb. nov. and Ruminococcus productus comb. nov.

The 16S ribosomal DNA sequences of representative members of the family Peptococcaceae were determined. The members of the family examined were divided into the following four phylogenetic groups: Peptococcus niger ATCC 27731T (T = type strain), the Sarcina-Peptostreptococcus anaerobius group, the ruminococcus-coprococcus group, and the peptostreptococcus group. Peptococcus niger, the type species of the family, was not related to other members of the family. Peptostreptococcus anaerobius ATCC 27337T, the type strain of the type species of the genus Peptostreptococcus, was closely related to Clostridium sordellii NCIB 10717T (level of sequence similarity, 85%). Sarcina ventriculi GIFU 7886, a spore-forming anaerobic gram-positive coccus, clustered with Clostridium perfringens ATCC 13124T at a similarity value of 91%. Members of the Sarcina-Peptostreptococcus anaerobius group clustered with clostridia at similarity values ranging from 85 to 91%. The type strains of Peptostreptococcus prevotii, Peptostreptococcus asaccharolyticus, Peptostreptococcus micros, and Peptostreptococcus magnus clustered at levels of sequence homology of 84 to 93%. This cluster was not included in the Peptococcus niger group or the Peptostreptococcus anaerobius group. Thus, these members of the genus Peptostreptococcus should be separated from the other members of the genus and also from members of the family Peptococcaceae. The sequence of Peptostreptococcus productus ATCC 27340T was different from the sequences of Peptostreptococcus anaerobius and Peptococcus niger. The sequence of Streptococcus hansenii ATCC 27752T, a strictly anaerobic strain, was different from the sequences of other streptococci; this strain clustered with Peptostreptococcus productus, coprococci, and ruminococci. Several phenotypic characteristics of Streptococcus hansenii ATCC 27752T were similar to characteristics of ruminococci.(ABSTRACT TRUNCATED AT 250 WORDS)

Anaerobiosis↗

Effect of inoculum, pH, and medium on the activity of ciprofloxacin against anaerobic bacteria.

The in vitro activity of ciprofloxacin against 30 Bacteroides fragilis group, 30 Clostridium, and 30 Peptococcaceae strains was determined by the agar dilution method in two different culture media at three pH and in three inoculum densities. In Wilkins-Chalgren agar the MICs for 90% of the strains were 0.12 micrograms/ml for B. fragilis, 0.5 micrograms/ml for Clostridium spp., and 2 micrograms/ml for Peptococcaceae. The pH and medium composition affected the MICs of ciprofloxacin.

Anti-Bacterial Agents↗

Comparative in vitro activity of 1-oxa-beta-lactam (LY127935) and cefoperazone with other beta-lactam antibiotics against anaerobic bacteria.

The in vitro activity of 1-oxa-beta-lactam (LY127935), cefoperazone (T-1551), cefuroxime, cefsulodin, cefaclor, cefotaxime, and cefoxitin on 85 anaerobic clinical isolates (30 Bacteroides, 30 Clostridium, 25 Peptococcaceae) was simultaneously determined by the agar dilution test in two different media, Brucella Agar (Difco Laboratories) and Wilkins-Chalgren agar. In Wilkins-Chalgren agar, 90% of Bacteroides were inhibited by (micrograms per milliliter): LY127935, 0.5; T-1551, 64; cefoxitin or cefuroxime, 8; cefsulodin or cefotaxime, 32; and cefaclor, 128. All Clostridia were inhibited in Wilkins-Chalgren by (micrograms per milliliter): LY127935, 4; T-1551, 2; cefoxitin, 6; cefuroxime, 0.12; cefsulodin, 0.5; cefaclor, 1; and cefotaxime, 8. All Peptococccaceae were inhibited by T-1551, cefsulodin or cefotaxime at 4 microgram/ml and by cefoxitin or cefuroxime at 1 to 2 microgram/ml. With cefaclor at 8 microgram/ml, 92% of strains were inhibited, and LY127935 at 16 microgram/ml only inhibited 64% of strains. LY127935 was the most active of the antibiotics tested against Bacteroides, showing good activity against Clostridia and poor activity on Peptococcaceae, whereas T-1551 was more active against Peptococccaceae and had similar activity against Clostridia and poor activity on Bacteroides. There are no significant differences between minimal inhibitory concentrations obtained in Brucella Agar and those obtained in Wilkins-Chalgren.

Anaerobiosis↗

Use of nonprotein nitrogen in pigs: effects of dietary urea on the intestinal microflora.

The fecal flora of 2 pigs given an optimal protein diet (digestive crude protein, 13.3%) with 2% urea, 2 pigs given a low protein diet (digestive crude protein, 5.7%) with 2% urea, and 2 pigs given a low protein diet only were bacteriologically investigated before feeding of each diet and on feeding days 21 and 38. The addition of urea to a low protein diet was significantly decreased Lactobacillus (P less than 0.001), Bacteroidaceae (P less than 0.01), Peptococcaceae (P less than 0.05), and Megasphaera (P less than 0.01) in the feces. In addition, there was a marked suppression of anaerobic curved rods (mainly Selenomonas) to undetectable amounts in the feces. A significant reduction (P less than 0.01) of Bifidobacterium and a marked suppression of Megasphaera to undetectable amounts were observed in the feces of pigs given low protein diets without urea. Microbial groups in the ileum and colon of pigs on feeding day 38 were also analyzed. Effects of dietary urea on the intestinal flora included: (i) anaerobic curved rods and spiral rods were restricted mainly to the colon, (ii) smaller numbers of yeasts were present in the intestine, and (iii) Bacteroidaceae, Peptococcaceae, and Eubacterium in the colon were not changed. Many anaerobic isolates, including curved rods and spiral rods, in the colon of pigs may have an important role in urea use.

Animals↗

Feasibility of exact species identification in routine diagnosis of anaerobic infections.

Gas-liquid chromatographic analysis of metabolic products and conventional biochemical tests are actually used for the identification of clinical isolates of anaerobes. As the majority of bacteremic and other life-threatening anaerobic infections are caused by Bacteroides fragilis, the prompt and accurate identification of this highly resistant species is essential for effective treatment. The clinical significance of the identification of the other Bacteroidaceae species and Peptococcaceae is discussed.

Anaerobiosis↗

Methodological implications of testing anaerobe susceptibility to cephalosporins (cefazolin, cefamandole, cefoxitin).

By simultaneously performing broth dilution, agar dilution, and agar diffusion tests with Bacteroidaceae and Peptococcaceae, the influence of methodology upon the outcome of susceptibility testing to cefazolin, cefamandole and cefoxitin was studied. With beta-lactamase positive and negative Bacteroidaceae, the results of the broth dilution and agar dilution tests were in good agreement for cefoxitin. However, when tested with cefaxolin and cefamandole, beta-lactamase positive Bacteroides strains had mostly high broth dilution MICs and relatively low agar dilution MICs. The statistical analysis of the relationship between zone size and broth dilution or agar dilution MICs frequently showed lack of stochastic linearity or relatively low correlation coefficients.

Bacteroidaceae↗

[Antibiotic resistance of anaerobic bacteria (author's transl)].

Reference data on the overall resistance patterns of anaerobic bacteria are needed since in individual clinical cases susceptibility testing is complicated by the long generation time and difficulties of test conditions. Benzylpenicillin and tetracyclines are the drugs of choice against Clostridium perfringens. Newer cephalosporins are also active, whereas other Clostridium species have been found resistant. Beta-lactam antibiotics show high activity against Peptococcaceae; resistance to clindamycin should be noted, however. The good in vitro activity of tetracyclines, clindamycin and erythromycin against Propionibacterium acnes could be confirmed in the systemic and local therapy of acne vulgaris. In contrast to gram-positive anaerobic bacteria, only some penicillin and cephalosporin derivatives are sufficiently active against gram-negative rods of the Bacteroides group. Sporadic resistance is seen to the newer tetrayclines, nitroimidazole compounds and to lincomycins. Nevertheless, these substances must be considered as standard therapeutics for infections caused by anaerobic gram-negative bacteria. It should be mentioned that cefoxitin may offer new therapeutic possibilities.

Actinomyces↗

Thermincola ferriacetica sp. nov., a new anaerobic, thermophilic, facultatively chemolithoautotrophic bacterium capable of dissimilatory Fe(III) reduction.

A moderately thermophilic, sporeforming bacterium able to reduce amorphous Fe(III)-hydroxide was isolated from ferric deposits of a terrestrial hydrothermal spring, Kunashir Island (Kurils), and designated as strain Z-0001. Cells of strain Z-0001 were straight, Gram-positive rods, slowly motile. Strain Z-0001 was found to be an obligate anaerobe. It grew in the temperature range from 45 to 70 degrees C with an optimum at 57-60 degrees C, in a pH range from 5.9 to 8.0 with an optimum at 7.0-7.2, and in NaCl concentration range 0-3.5% with an optimum at 0%. Molecular hydrogen, acetate, peptone, yeast and beef extracts, glycogen, glycolate, pyruvate, betaine, choline, N-acetyl-D-glucosamine and casamino acids were used as energy substrates for growth in presence of Fe(III) as an electron acceptor. Sugars did not support growth. Magnetite, Mn(IV) and anthraquinone-2,6-disulfonate served as the alternative electron acceptors, supporting the growth of isolate Z-0001 with acetate as electron donor. Formation of magnetite was observed when amorphous Fe(III) hydroxide was used as electron acceptor. Yeast extract, if added, stimulated growth, but was not required. Isolate Z-0001 was able to grow chemolithoautotrophicaly with molecular hydrogen as the only energy substrate, Fe(III) as electron acceptor and CO(2) as the carbon source. Isolate Z-0001 was able to grow with 100% CO as the sole energy source, producing H(2) and CO(2), requiring the presence of 0.2 g l(-1) of acetate as the carbon source. The G+C content of strain Z-0001(T )DNA G+C was 47.8 mol%. Based on 16S rRNA sequence analyses strain Z-0001 fell into the cluster of family Peptococcaceae, within the low G+C content Gram-Positive bacteria, clustering with Thermincola carboxydophila (98% similarity). DNA-DNA hybridization with T. carboxydophila was 27%. On the basis of physiological and phylogenetic data it is proposed that strain Z-0001(T) (=DSMZ 14005, VKM B-2307) should be placed in the genus Thermincola as a new species Thermincola ferriacetica sp. nov.

Acetates↗

A reassessment of the microbial flora of the female genital tract, with special reference to the occurrence of Bacteroides species.

Two hundred and twelve randomly selected vaginal or uterine cervical specimens were investigated for the presence of anaerobic and aerobic bacteria and yeasts. Anaerobes of possible clinical significance, including Bacteroidaceae, Peptococcaceae and clostridia were isolated from 34% of the specimens and were identified to specific or generic level. Among the Bacteroidaceae isolated, B. bivius was the most common, followed by other propionate-negative species. Members of the Bacteroides fragilis group were seldom isolated. Of the aerobic or facultatively anaerobic isolates, enterococci and Escherichia coli were most often found. The results show that clinically significant anaerobes, especially Bacteroides species, are not regular members of the vaginal flora and that the species distribution of anaerobes occurring in the genital tract is significantly different from that of the intestinal tract.

Bacteria↗

Thermincola carboxydiphila gen. nov., sp. nov., a novel anaerobic, carboxydotrophic, hydrogenogenic bacterium from a hot spring of the Lake Baikal area.

A novel anaerobic, thermophilic, alkalitolerant bacterium, strain 2204(T), was isolated from a hot spring of the Baikal Lake region. The cells of strain 2204(T) were straight rods of variable length, Gram-positive with an S-layer, motile with one to two lateral flagella, and often formed aggregates of 3-15 cells. The isolate was shown to be an obligate anaerobe oxidizing CO and producing equimolar quantities of H(2) and CO(2) according to the equation CO+H(2)O-->CO(2)+H(2). No organic substrates were used as energy sources. For lithotrophic growth on CO, 0.2 g acetate or yeast extract l(-1) was required but did not support growth in the absence of CO. Growth was observed in the temperature range 37-68 degrees C, the optimum being 55 degrees C. The pH range for growth was 6.7-9.5, the optimum pH being 8.0. The generation time under optimal conditions was 1.3 h. The DNA G+C content was 45 mol%. Penicillin, erythromycin, streptomycin, rifampicin, vancomycin and tetracycline completely inhibited both growth and CO utilization by strain 2204(T). Thus, isolate 2204(T) was found to be the first known moderately thermophilic and alkalitolerant H(2)-producing anaerobic carboxydotroph. The novel bacterium fell within the cluster of the family Peptococcaceae within the low-G+C-content Gram-positive bacteria, where it formed a separate branch. On the basis of morphological, physiological and phylogenetic features, strain 2204(T) should be assigned to a novel genus and species, for which the name Thermincola carboxydiphila gen. nov., sp. nov. is proposed. The type strain is strain 2204(T) (=DSM 17129(T)=VKM B-2283(T)=JCM 13258(T)).

Anti-Bacterial Agents↗

Utilization of ammonia nitrogen by intestinal bacteria isolated from pigs.

In a medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, 17 of 24 Bacteroidaceae strains, 3 of Selenomonas strains, 1 of 7 curved rods, 3 of 7 Spirochaetaceae strains, 8 of 20 Eubacterium strains, 8 of 13 Peptococcaceae strains, 3 of 4 Clostridium strains, 19 of 20 Enterobacteriaceae strains, and 1 of 8 Streptococcus strains utilized ammonia nitrogen preferentially to proteose peptone nitrogen. To determine the ability of intestinal microbes to synthesize amino acids from ammonia, ammonia utilization by Bacteroides ruminicola strain 9 was studied in defined media containing ammonia and other nitrogen sources. In another medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, ammonia was preferentially utilized even when the proteose peptone nitrogen content was eight times greater than that of ammonia nitrogen. In a medium containing ammonia, an amino acid, and cysteine, the lowest uptake of ammonia nitrogen was observed when the medium contained aspartic acid, glutamic acid, threonine, or alanine; but ammonia was utilized more effectively than any of the amino acids. Incorporation of (15)N from [(15)N]ammonia into bacterial amino acids was studied. (15)N was incorporated into every amino acid of B. ruminicola strain 9, and the highest uptake was observed in aspartic acid and alanine.

Amino Acids↗

Establishment of specific pathogen-free guinea-pig colonies using limited-flora guinea-pigs associated with conventional guinea-pig flora, and monitoring of their cecal flora.

Six groups of limited flora (LF) Hartley guinea-pigs were produced by inoculation of hysterectomy-derived GF guinea-pigs with various combinations of cecal bacteria of conventional (CV) guinea-pigs to determine the effective bacterial cocktails for the establishment of a specific pathogen free (SPF) colony. Bifidobacterium magnum (Bif) isolated from CV guinea-pigs was used for pretreatment. The mortality of LF guinea-pigs inoculated with only Bif was 75%, and that of those inoculated with Bif plus chloroform-treated cecal suspension (CHF) or Bif plus CHF plus 32 isolates from CV guinea-pigs was 40 to 66.7%. These three groups were in an unhealthy condition with mucoid enteritis-like diarrhea. However, the mortality of LF guinea-pigs inoculated with the anaerobic growth on EG plates injected with 10(-5) dilution of cecal contents (CF) or inoculated with Bif plus CF was 6.3 and 15%, respectively. These latter two groups of LF guinea-pigs were transferred to separate barrier rooms and some of the LF guinea-pigs were maintained in isolators as a source of intestinal flora for SPF guinea-pigs. The composition of cecal flora of LF guinea-pigs was stable for a long time, and bacteroidaceae and peptococcaceae were maintained as predominant components. The basic composition of the cecal flora of SPF guinea-pigs originated from LF guinea-pigs, which consists mainly of the anaerobic bacteria, was not changed over a long period, and the flora composition became similar to that in CV guinea-pigs. Guinea-pig-specific pathogens from the SPF colonies were not detected during experiments.

Animals↗

In vitro activity of tiamulin (81.723 HFU), a new pleuromulin derivative, against clinically significant anaerobes.

The susceptibility of more than 40 strains of Gram-negative and Gram-positive anaerobes to tiamulin (Sandoz 81.723 hfu), a new pleuromulin (pleuromutilin) derivative, was determined by broth dilution and agar dilution tests. The influences of density of the inoculum upon MICs was studied by a specially designed pour plate-technique. Bacteroides fragilis, B. vulgatus, B. splanchnicus, B. oralis, B. asaccharolyticus, B. melaninogenicus, Fusobacterium fusiforme (F. nucleatum), Sphaerophorus necrophorus, Clostridium perfringens, C. fallax, Propionibacterium acnes and several species of Peptococcaceae showed broth dilution MICs of 0.03 similar to 1 microgram/ml. Members of B. thetaiotaomicron, B. distasonis and S. freundii (F. mortiferum) were inhibited by 8 similar to 32 microgram/ml and 2 strains of S. varius had a broth dilution MIC of 256 microgram/ml. With most strains, the agar dilution MICs were 2 similar to 4 similar to 8 times the broth dilution MICs. In pour plate-tests, the MICs were not considerably influenced influenced by varying initial concentrations of viable cells. With most anaerobes, the MBCs of tiamulin were more than 100-fold higher than the MICs. The results obtained indicated that, apart from S. varius, B. thetaiotaomicron, B. distasonis and S. freundii (F. mortiferum), members of 16 other anaerobic species including B. fragilis were without exception sensitive to tiamulin.

Anti-Bacterial Agents↗

In vitro activity of flomoxef compared to moxalactam, cefoxitin, cefotaxime, and clindamycin against anaerobes.

To assess the in vitro activity of flomoxef (6315-S), moxalactam, cefoxitin, cefotaxime, and clindamycin against anaerobes 197 clinical isolates (27 Bacteroides fragilis, 42 B. thetaiotaomicron, 10 B. vulgatus, 7 B. ovatus, 6 B. uniformis, 6 B. distasonis, 7 Bacteroides melaninogenicus group, 11 Bacteroides oralis group, 21 Clostridium difficile, 7 C. perfringens, 3 C. sporogenes, 3 Clostridium spp., 33 Propionibacterium acnes, 14 Peptococcaceae) were studied by means of agar dilution tests. The MIC90 of B. fragilis was less than 2 micrograms/ml for flomoxef, less than 4 micrograms/ml for moxalactam, less than 16 micrograms/ml for cefoxitin, less than 128 micrograms/ml for cefotaxime and less than 2 micrograms/ml for clindamycin. The respective MIC90's of B. thetaiotaomicron were less than 64, less than 128, less than 32, less than 256 and 8 micrograms/ml. Strains of the other Bacteroides species and groups were more susceptible to flomoxef and the other antibiotics than B. thetaiotaomicron. Against Clostridium difficile flomoxef (MIC90 less than 4 micrograms/ml) proved to be superior to the other agents tested. Most of the Clostridium strains other than C. difficile were also susceptible to flomoxef; anaerobic grampositive cocci and Propionibacterium acnes were very sensitive (MIC90's less than 1 and less than or equal to 0.125 micrograms/ml, respectively). Its anti-anaerobic activity, together with its efficacy against aerobes, should make flomoxef a useful adjunct to the arsenal of modern antibiotic therapy.

Anti-Bacterial Agents↗

[Susceptibility to Thiamphenicol and Chloramphenicol of Anaerobic Bacteria (author's transl)].

The in vitro susceptibility to thiamphenicol and chloramphenicol of 272 anaerobes, most of which were recent clinical isolated, was determined by broth dilution tests, With chloramphenicol, 133 anaerobic gram-negative non-sporing rods (48 Bacteroides fragilis, 13 B. thetaiotaomicron, 14 B. oralis, 16 Sphaerophorus varius etc.) had MIC values of 0.03 through 16 microng/ml. Very similar results (MIC, 0.06-16 microng/ml) were obtained with thiamphenicol. In concentrations of 4 microng/ml or less chloramphenicol inhibited 90.2% and thiamphenicol 78.95% of the strains. Strains with only moderate sensitivity to both antibiotics (MIC, 8 or 16 microng/ml) belonged to B. fragilis or other Bacteroides species. Members of the Fusobacterium and Sphaerophorus group were susceptible to less than or equal to 2 microng chloramphenicol/ml and less than or equal to 4 microng thiamphenicol/ml respectively. With both antibiotics, 102 strains of gram-positive non-sporing anaerobes (P. acnes, Peptostreptococcus spp., Peptococcus spp.) were susceptible to less than or equal to 8 microng/ml. Of 37 Clostridium isolates, 35 (belonging to C. perfringens, C. septicum, C. cadaveris etc.) were inhibited by concentrations of 8 microng/ml or less of chloramphenicol and thiamphenicol. Only one strain each of C. perfringens and Clostridium sp. had an MIC of 16 microng thiamphenicol/ml. Accordingly, resistance to thiamphenicol or chloramphenicol was not observed. A standardized monodisk agardiffusion test was performed on 40 Bacteroidaceae, 18 Peptococcaceae and 20 C, (P.) acnes strains. Only a poor correlation was observed between MIC and zone size for thiamphenicol and chloramphenicol. Therefore, inhibition zone diameter measurement cannot be regarded as a reliable method to detect chloramphenicol or thiamphenicol resistance in anaerobes. Thiamphenicol, which is virtually as active against anaerobes as chloramphenicol but lacks serious toxicity, may well play an important role in the therapy of various anaerobic infections.

Anti-Bacterial Agents↗