Evaluation of a new chromogenic medium for the isolation and presumptive identification of Salmonella species from stool specimens.

The performance of BBL CHROMagar Salmonella (Becton Dickinson, France), a new selective chromogenic medium for the isolation and presumptive identification of Salmonella spp., was evaluated. On this medium, which is a modification of CHROMagar Salmonella (CHROMagar Microbiology, France) with enhanced selectivity, the colonies of Salmonella are stained in mauve (rose-violet), while those of other organisms appear in blue-green or are not stained by any of the chromogens of the medium. The medium was evaluated with a total of 176 strains of Salmonella and other organisms, consisting of 18 reference strains and 158 clinical isolates. All Salmonella strains except subspecies IIIa and IIIb strains and Salmonella Gallinarum yielded typical mauve colonies. During the evaluation with 107 known positive and 332 unknown stool specimens in a clinical laboratory, a total of 115 and 105 Salmonella isolates were obtained on BBL CHROMagar Salmonella and Hektoen enteric agar, respectively. From the known positive stool specimens, 92 true positive cultures were obtained on BBL CHROMagar Salmonella and 89 on Hektoen enteric agar, yielding sensitivities of 86 and 83%, respectively. From the unknown stool specimens, a total of 27 Salmonella isolates were obtained, with 23 isolated from BBL CHROMagar Salmonella and 16 from Hektoen enteric agar by direct plating (sensitivity 85 and 59%, specificity 99 and 97%, respectively). Seroagglutination tests could be performed directly from BBL CHROMagar Salmonella. Compared to conventional isolation media, the time needed for confirmatory biochemical and serological tests was shortened by about 1 day when BBL CHROMagar Salmonella was used. On the basis of these results, the medium can be recommended for the primary isolation and presumptive identification of Salmonella spp. from clinical stool specimens.

Key enzymes for the degradation of benzoate, m- and p-hydroxybenzoate by some members of the order Actinomycetales.

A preliminary screening of numerous species of the order Actinomycetales, especially of the genera Mycobacterium, Nocardia, Rhodococcus, Pseudonocardia, and Streptomyces, showed that many of them are able to metabolize benzoate (B) and p-hydroxybenzoate (pHB) as indicated by growth and change of color of the pH-indicator of an agar medium. Subsequent experiments with liquid cultures which allowed the analysis of substrate utilization by thin layer chromatography confirmed these results. The study of the degradative pathway proved that B was metabolized via catechol (C), pHB via protocatechuate (P) and m-hydroxybenzoate (mHB) via gentisate (G). The aromatic ring of C and P was subjected to an ortho-cleavage; only one strain of Noc. asteroides degraded C via a meta-cleavage, but P via an ortho-cleavage. Cell free extracts of four selected organisms exhibited activity of C-1,2-dioxygenase (C-1,2-O) and/or P-3,4-dioxygenase (P-3,4-O), depending on the growth substrate used for precultivation. In Streptomyces C-1,2-O was only found in cells grown on B, and P-3,4-O only in cells grown on pHB. On the contrary, in Rhodococcus rhodochrous B-cells oxidized C as well as P, while P-cells possessed only P-3,4-O-activity.

Evaluation of BBL CHROMagar orientation medium for detection and presumptive identification of urinary tract pathogens.

The microbiological performance of BBL CHROMagar Orientation medium and CPS ID2 agar was compared to that of Columbia agar with 5% sheep blood and MacConkey agar without crystal violet for the enumeration and presumptive identification of bacteria responsible for urinary tract infections. Of a total of 658 clinical urine specimens, 118 specimens yielded no growth, 402 specimens yielded growth with cell counts of > or = 10(5) CFU/ml, and 138 specimens yielded growth with cell counts of < 10(5) CFU/ml. Of the specimens with cell counts of > or = 10(5) CFU/ml, 163 were pure cultures and 239 were mixed cultures. A total of 266 Escherichia coli organisms were isolated on both chromogenic media, 260 were isolated on blood agar, and 248 were isolated on MacConkey agar. One strain (0.4%) failed to develop the expected pink color on CHROMagar Orientation medium, and 23 strains (8.7%) failed to develop the expected pink color on CPS ID2 agar. Enterococci (CHROMagar Orientation medium, n = 266; CPS ID2 agar, n = 265) produced small blue-green colonies on both chromogenic media. Fifty of the mixed cultures contained enterococci that were detected only on the chromogenic media. The Klebsiella-Enterobacter-Serratia (KES) and the Proteus-Morganella-Providencia (PMP) groups could be identified on both chromogenic media. Of 66 isolates of the KES group, 63 grew with the expected color on CHROMagar Orientation medium and 58 of 64 isolates grew with the expected color on CPS ID2 agar. Other microorganisms required further identification. The use of chromogenic medium formulations offers a time-saving method for the reliable detection, enumeration, and presumptive identification of urinary tract pathogens. One of the greatest advantages of these media is the easy recognition of mixed cultures.

Endocarditis caused by a Leptotrichia buccalis-like bacterium in a patient with a prosthetic aortic valve.

The isolation of a Leptotrichia buccalis-like bacterium from the blood of a patient with endocarditis associated with a prosthetic aortic valve is reported. The organism was isolated from different blood cultures. It was similar but not completely identical to Leptotrichia buccalis. The microbiological identification was confirmed independently by three laboratories, including two reference institutions. The case and the characteristics of the microorganism are reported.

Characterization of NAD-dependent 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and of NADP-dependent 7 beta-hydroxysteroid dehydrogenase from Peptostreptococcus productus.

A human fecal isolate, characterized by morphological, physiological and biochemical data as a strain of Peptostreptococcus roductus, was shown to contain NAD-dependent 3 alpha- and 3 beta-hydroxysteroid dehydrogenases and a NADP-dependent 7 beta-hydroxysteroid dehydrogenase. All enzyme activities could be demonstrated in crude extracts and in membrane fractions. The 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were synthesized constitutively. Specific enzymatic activities were significantly reduced when bacteria were grown in the presence of 3-keto bile acids, while other bile acids were ineffective. For the 3 alpha (3 beta)-hydroxysteroid dehydrogenase, a pH optimum of 8.5 (9.5) and a molecular weight of 95,000 (132,000) was estimated. 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were heat-sensitive (about 75% inactivation at 50 degrees C for 10 min). The 7 beta-hydroxysteroid dehydrogenase was already present in uninduced cells, but specific activity could be enhanced up to more than 2.5-fold when bacteria were grown in the presence of 7-keto bile acids. Disubstituted bile acids were more effective than trisubstituted ones, ursodeoxycholic acid was ineffective as an inducer. A pH optimum of 10.0 and a molecular weight of about 82,000 were shown for the 7 beta-hydroxysteroid dehydrogenase. The enzyme preparation reduced the 7-keto group of corresponding bile acids. Again the affinities of disubstituted bile acids for the enzyme were higher than those of the trisubstituted bile acids, but no significant differences between conjugated and free bile acids were observed. The 7 beta-hydroxysteroid dehydrogenase was heat-sensitive (72% inactivation at 50 degrees C for 10 min), but was detectable at 4 degrees C for at least 48 h.

Application of non-radioactive BACTEC system to sterility testing and hygiene.

The applicability of the non-radioactive BACTEC NR 660 system for the detection of low viable counts of aerobic and anaerobic bacteria (including non-fermenters and aerobic spore-forming bacteria) and fungi was investigated. The BACTEC system together with the usual BACTEC NR 6A and NR 7A media was able to detect most of the organisms without supplementation of the media; for growth of lactobacilli and other acidophilic organisms, sterile orange juice was used as a supplement. Microbial growth was detected from pure cultures and also from artificially contaminated beverages and ointment bases. Detection times varied according to the types of metabolism of the respective strains and their initial viable counts, but usually they were detected after 20 to 42 h of incubation. The BACTEC NR 660 system seems to be applicable not only for blood cultures in medical microbiology but also in pharmaceutical and food industry.

Newly recovered and delineated microbial species of the human genital tract.

Several new genitourinary Bacteroides species and the new Mobiluncus/Falcivibrio have been described since 1977. The normal vaginal flora consists mainly of lactobacilli and other "non-pathogenic" aerobes and anaerobes. Under unknown circumstances, organisms may increase in number, displacing the normal lactobacillus flora and raising the vaginal pH. This renders a more favourable biotope for organisms with a pH optimum in the neutral or alkaline range. Overgrowth of Gardnerella, Bacteroides, B-streptococci and Mobiluncus/Falcivibrio is indicative of vaginosis, which is not a typical sexual disease, but may be sexually transmitted. Proper treatment includes the male partner. No overall-valid therapy exists, but metronidazole is the drug of choice if Bacteroides or Gardnerella form the disturbed flora, and beta-lactams are the drugs of choice when Mobiluncus or aerobes are involved.

NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities from a lecithinase-lipase-negative Clostridium species 25.11.c.

A lecithinase-lipase-negative Clostridium sp. 25.11.c., not fitting in any of the species of Clostridia described so far as judged by morphological, physiological, and biochemical data, was shown to contain NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenases. The three hydroxysteroid dehydrogenases could be demonstrated in the supernatant and in the membrane fraction after solubilization with Triton X-100, suggesting enzymes which were originally membrane bound. The 3 beta-hydroxysteroid dehydrogenase was synthesized constitutively, and the specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids and trisubstituted bile acids. A pH optimum of 7.5 and a molecular weight of approx. 104,000 were estimated by molecular sieve chromatography. The enzyme reduced the 3-keto group of bile acids; an oxidation of a 3 beta-hydroxyl function could not be demonstrated. The lowest Km values were found for disubstituted bile acids, trisubstituted and conjugated bile acids having higher Km values. 7 alpha-Hydroxysteroid dehydrogenase, but not 7 beta-hydroxysteroid dehydrogenase, was already present in uninduced cells. The specific activities, however, were greatly enhanced when cells were grown in the presence of chenodeoxycholic acid or 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid. Ursodeoxycholic acid with its 7 beta-hydroxyl group was ineffective as an inducer. Molecular weights of approx. 82,000 and 115,000 were found for the 7 alpha-hydroxysteroid dehydrogenase and the 7 beta-hydroxysteroid dehydrogenase, respectively. In contrast to the in vivo situation, the reaction could only be demonstrated in the reductive direction in vitro. Here, the pH optimum for the overall reaction was 8.5-8.7. 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities were readily demonstrated for at least 48 h when preparations were stored at 4 degrees C, but were found to be heat-sensitive.

Quantitative studies on the vaginal flora of asymptomatic women and patients with vaginitis and vaginosis.

Vaginal washings of 22 patients with vaginitis, 11 with vaginosis, and 12 healthy subjects were investigated quantitatively and qualitatively for aerobic and anaerobic bacteria and yeasts. Gardnerella vaginalis was recovered from 9 of the vaginitis patients, 7 of the vaginosis patients, and 4 of the asymptomatic subjects. Obligate anaerobes were found in 11 of the vaginitis patients, 4 of the vaginosis patients, and none of the control subjects. Bacteroides bivius was the anaerobe most frequently isolated from symptomatic subjects. Anaerobic vibrios were recovered twice from symptomatic subjects. The counts for Gardnerella vaginalis and anaerobes when present were generally very high. The most frequent aerobes were beta-hemolytic streptococci (group B) and staphylococci.

Isolation, characterization and ecological distribution of recently recognized propionic acid-negative Bacteroides species from human sources.

140 strains of anaerobic, Gram-negative, propionate-negative, non-pigmented rods (Bacteroides oralis group) were isolated from normal flora and from clinical specimens. They were characterized by physiological tests, gas chromatography of the acid end products, and determination of the G + C content of their DNA. In addition, their distribution in the various biotopes of the human body (oropharyngeal tract, genitourinary tract, and gut) and in clinical specimens was investigated. The strains could be assigned to the recently described species Bacteroides denticola, B. buccae, B. buccalis, and B. oris, or to B. bivius, B. disiens and B. oralis. In the investigated group, especially B. bivius exhibits an antibiotic resistance--particularly against the beta-lactam antibiotics--comparable to that of B. fragilis.

A newly discovered sialidase from Gardnerella vaginalis.

A sialidase (neuraminidase, acylneuraminosyl hydrolase, EC 3.2.1.18) has been discovered and isolated from Gardnerella vaginalis (ex. Haemophilus vaginalis), a possibly pathogenic inhabitant of the female genital tract. Bacteria were grown in peptone-yeast-extract medium with 2.0 mM N-acetylmannosamine as enzyme inductor under CO2 atmosphere. Sialidase activity was found in the bacterial sediment and in the culture medium. The enzyme was liberated from the cells by ultrasonic treatment. Purification was performed by 60-80% ammonium sulfate precipitation and by column chromatography on Sepharose CL-6B and Sephadex G 200. The enzyme revealed a molecular weight in the range of Mr 75 000 and a pH optimum at 5.5. Among the different types of NeuAc-containing glycoconjugates, the enzyme exhibits its highest activities towards the globular glycoproteins alpha 1-acid glycoprotein and fetuin. Taking their cleavage rate as 100, it is around 55 for II3NeuAc-Lac, 45 for bovine submaxillary mucin, 35 for II6NeuAc-Lac and IV3, III6NeuAc2-LcOse4. The rates for III8,II3NeuAc2-Lac, gangliosides and colominic acid are below 20. Due to its specificity pattern, the enzyme may play a role in the pathogenic process of G. vaginalis infections.

[Role of Gardnerella vaginalis and anaerobes. Etiology of nonspecific colpitis]. / Die Rolle von Gardnerella vaginalis und Anaerobiern. Atiologie der unspezifischen Kolpitis.

Nonspecific vaginitis is a very common clinical syndrome. In patients with nonspecific vaginitis the normal, lactobacillus-dominated microbial flora is replaced by Gardnerella vaginalis and certain anaerobes, preferably bacteroides spp. The mechanisms by which anaerobes or Gardnerella vaginalis or a combination of these organisms produce the disease condition are still unknown. Treatment with ampicillin and certain other drugs is often effective, but metronidazole appears to be the most effective antimicrobial for this condition. The optimal dose and duration of metronidazole therapy require further study.

[Efficacy of tinidazole against anaerobes in comparison with metronidazole, ornidazole, cefoxitin and lamoxactam]. / Die Anaerobierwirksamkeit von Tinidazol im Vergleich mit Metronidazol, Ornidazol, Cefoxitin und Lamoxactam.

The in vitro inhibitory activity of tinidazole, metronidazole, ornidazole, cefoxitin and moxalactam was determined against 150 isolates of clinically important anaerobes including Bacteroides fragilis, Bacteroides bivius and Clostridium perfringens by means of agar dilution tests. The members of 18 gramnegative and 14 grampositive species were inhibited by tinidazole at less than or equal to 0,01-8 micrograms/ml thus being without exception susceptible to the drug. A similar in vitro activity was recorded for metronidazole and ornidazole. Though cefoxitin and moxalactam generally had a good in vitro activity against most anaerobic species, single strains of Bacteroides thetaiotaomicron, B. distasonis, Clostridium difficile and Eubacterium rectale were resistant to these drugs. With regard to its in vitro activity, tinidazole seems to be a promising substance for the therapy of anaerobic infections.

[Anaerobic effect of tinidazol compared with metronidazol, ornidazol, cefoxitin and lamoxactam]. / Die Anaerobierwirksamkeit von Tinidazol im Vergleich mit Metronidazol, Ornidazol, Cefoxitin und Lamoxactam.

The in vitro inhibitory activity of tinidazole, metronidazole, ornidazole, cefoxitin and moxalactam was determined against 150 isolates of clinically important anaerobes including Bacteroides fragilis, Bacteroides bivius and Clostridium perfringens by means of agar dilution tests. The members of 18 gramnegative and 14 grampositive species were inhibited by tinidazole at less than or equal to 0,01-8 micrograms/ml thus being without exception susceptible to the drug. A similar in vitro activity was recorded for metronidazole and ornidazole. Though cefoxitin and moxalactam generally had a good in vitro activity against most anaerobic species, single strains of Bacteroides thetaiotaomicron, B. distasonis, Clostridium difficile and Eubacterium rectale were resistant to these drugs. With regard to its in vitro activity, tinidazole seems to be a promising substance for the therapy of anaerobic infections.

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.

[CO2-dependent mutant of Salmonella typhi causing typhoid fever in two patients]. / CO2-abhängige Mutante von Salmonella typhi als Ursache typhöser Erkrankungen bei zwei Patienten.

A CO2-dependent mutant of Salmonella typhi was isolated from anaerobic but not from aerobic blood-cultures of two patients with typhoid fever. Both patients diseased after return from their home country Turkey. The strains developed in a CO2-enriched atmosphere and under anaerobic but not aerobic culture conditions. This property remained stable during 20 subcultures. The fermentative and serological pattern was identical with S. typhi T2. Both strains were of lysovar E1a.

Presence of diaminopimelic acid in propionate-negative Bacteroides species and in some butyric acid-producing strains.

AThe presence of diaminopimelic acid (m-DAP) in strains of Bacteroides melaninogenicus, B. bivius and other species as well as in unidentified strains of Bacteroides was investigated by thin-layer chromatography. Strains of B. bivius and B. disiens all contained m-DAP as did the subspecies intermedius and melaninogenicus of B. melaninogenicus. Strains of B. asaccharolyticus and similar black pigment-producing butyrate-positive isolates showed heterogeneity. Asaccharolytic strains were DAP negative, whereas two strains fermenting glucose were positive. Some of the non-pigmented propionate-negative and butyrate-negative unidentified strains also contained DAP. The consistent finding of m-DAP in strains of B. bivius, B. disiens, and B. melaninogenicus indicates that DAP detection might be of value in the identification of these species.

Neuraminidases of Bacteroidaceae die neuraminidasen von bacteroidaceen.

Neuraminidases can be detected in members of the anaerobic gram-negative non-sporing rods (Bacteroidaceae), especially in the genus Bacteroides. B. fragilis, the most virulent species, has the highest neuraminidase activity, while the other intestinal species exhibit markedly lower activities or the enzyme is completely absent. Members of the Bacteroides oralis group, so far investigated, degrade only substrates of lower molecular weight.

Thin layer chromatography for rapid detection of carbohydrate utilization by Bacteroides strains.

19 weakly saccharolytic Bacteroides strains of different species were tested by thin-layer chromatography (TLC) for their fermentative abilities for fructose, sucrose, lactose, galactose, and raffinose. Conventional fermentation tests were run parallel. In general, a good agreement between both methods was recorded. Two strains, however, showed a degradation in the TLC test without an acidification. With some strains, sucrose as a substrate yielded a fructose spot, lactose a galactose spot, and raffinose a melibiose spot, indicating an incomplete degradation of these carbohydates.