Simplified procedure for the routine isolation of Clostridium difficile from faeces.

The use of alcohol, at a final concentration of 50%, as a selective procedure for the isolation of Clostridium difficile was compared to a selective medium containing 250 microgram /ml of cycloserine and 10 microgram /ml of cefoxitin. Of 266 faecal samples 82 were shown to be positive by one or other method. Seventy-seven (94%) of these were detected by the selective agar (SA) and 72 (88%) by the alcohol procedure (AP). Ten samples (12%) were positive only by SA and five samples (6%) by AP only. The AP was further modified so that all manipulations prior to incubation were performed on the open bench. Of 18 positive samples, 18 (100%) were detected by SA and 16 (89%) by AP.     

Radiometric detection of mycobacteria in routine blood cultures.

Mycobacterium chelonei subsp. abscessus was detected radiometrically in routine blood cultures. The organism was detected in two patients without the use of special mycobacteriological media.     

Optimal time for routine early subculture of blood cultures.

Routine aerobic subcultures performed between 6 and 12 h and between 12 and 17 h after blood collection yielded 36 and 63%, respectively, of isolates from blood cultures, whereas only 10% of isolates were recovered in a preliminary study when cultures were subcultured within 6 h of blood collection for cultures.     

The possible significance of Clostridium spp. in blood cultures

The significance of Clostridium spp. in blood cultures was evaluated by two methods. In the first part of the study, a group of 80 patients with Clostridium spp. bacteraemia was compared with a group of 100 patients with Bacillus spp. in blood cultures, making the assumption that Bacillus almost invariably represents contamination (pseudobacteraemia). Significant differences were found between the two groups, suggesting that growth of Clostridium did not represent pseudobacteraemia. Patients with Clostridium bacteraemia were older, had a higher frequency of gastrointestinal disease (especially colorectal tumours), were associated more frequently with polymicrobial bacteraemia, and had a higher mortality rate. In the second part of the study, each of the 80 cases of Clostridium bacteraemia was evaluated individually for clinical relevance by an infectious diseases expert. In two-thirds of the cases, isolates of Clostridium from blood were considered to be of clinical relevance, whereas in one-third of cases, the clinical significance of this finding was doubtful. It was concluded that growth of Clostridium spp. in blood cultures, even in the absence of one of the histotoxic syndromes, is often of clinical significance, and that such findings should be properly evaluated and not ignored.     

Direct isolation of Candida spp. from blood cultures on the chromogenic medium CHROMagar Candida

CHROMagar Candida is a selective and differential chromogenic medium that has been shown to be useful for identification of Candida albicans, Candida krusei, Candida tropicalis, and perhaps Candida glabrata. Colony morphology and color have been well defined when CHROMagar Candida has been used to isolate yeast directly from clinical specimens, including stool, urine, respiratory, vaginal, oropharyngeal, and esophageal sources. Direct isolation of yeast on CHROMagar Candida from blood cultures has not been evaluated. We evaluated whether the color and colony characteristics produced by Candida spp. on CHROMagar Candida were altered when yeasts were isolated directly from blood cultures. Fifty clinical isolates of Candida were inoculated into aerobic and anaerobic blood culture bottles and incubated at 35 degrees C in an automated blood culture system. When growth was detected, an aliquot was removed and plated onto CHROMagar Candida. As a control, CHROMagar Candida plates were inoculated with the same isolate of yeast grown on Sabouraud dextrose agar simultaneously. No significant difference was detected in color or colony morphology between the blood and control isolates in any of the tested organisms. All C. albicans (n = 12), C. tropicalis (n = 12), C. glabrata (n = 9), and C. krusei (n = 5) isolates exhibited the expected species-specific colony characteristics and color, whether isolated directly from blood or from control cultures. CHROMagar Candida can be reliably used for direct isolation of yeast from blood cultures. Direct isolation could allow mycology laboratories to more rapidly identify Candida spp., enable clinicians to more quickly make antifungal agent selections, and potentially decrease patient morbidity and mortality.     

Effect of aerobic and anaerobic atmospheres on isolation of organisms from blood cultures.

Blood was cultured in two vacuum bottles containing Columbia broth. Filtered air was admitted to one bottle (aerobic); the unvented bottle was considered anaerobic. Cultures were incubated at 35 C until growth occurred or for at least 7 days. Of 744 organisms isolated, 50% were isolated from both bottles, 30% from the aerobic bottle only, and 20% from the anaerobic bottle only. These results indicate the need for use of both an aerobic and anaerobic bottle for blood cultures.     

Lack of clinical relevance in routine terminal subculturing of blood cultures.

The usefulness of performing final blind subcultures of previously negative blood cultures was evaluated over a 21-month period. From over 14,000 blood culture bottles blindly subcultured after 7 days of incubation, only 12 potentially significant organisms were found. The finding of these 12 organisms did not influence patient care since in 11 instances the same organism had already been reported from prior positive bottles and in 1 instance the patient had already died. These results suggest that blind 7-day subcultures are of minimal value. Other factors that need to be considered before eliminating the final subculture are presented.     

Pathology of Clostridium welchii type D enterotoxaemia. 3. Basis of the hyperglycaemic response

It has been demonstrated that the hyperglycaemia, which is a prominent feature of Clostridium welchii type D epsilon toxin intoxication, results from rapid mobilization of hepatic glycogen.Liver glycogen stores are rapidly depleted in intoxicated animals and no elevation of blood glucose level occurs in animals in which hepatic glycogen has been depleted prior to intoxication.No detectable interference with tissue respiration could be demonstrated in tissues from intoxicated animals, suggesting that the hyperglycaemia is not the result of interference with glycolysis.     

Evaluation of the necessity for routine terminal subcultures of previously negative blood cultures.

It has been recommended that blood cultures be routinely subcultured aerobically on the day after the specimen is received, anaerobically after 48 h, and, finally, after 5 to 7 days if the cultures appear negative (Bartlett et al., Cumitech 1, American Society for Microbiology, Washington, D.C., 1974). To evaluate the necessity for the final routine subculture, 2,780 previously negative blood culture bottles were subcultured after 7 days of incubation. Of four bottles positive by subculture, three yielded the same organism as previously isolated from the companion bottle, and one yielded an organism considered to represent a contaminant. Since the routine 7-day subculture did not significantly increase the yield from previously negative blood cultures, the time and expense of the terminal subculture appears not to be warranted. Whereas a total of 7 days of incubation of blood cultures is probably adequate for general hospitals, a second week of incubation appears indicated in selected cases of suspected endocarditis and persistent or recurrent infection, as well as in any referral center. Candida and fastidious gram-negative bacilli, such as Haemophilus, Cardiobacterium, and Actinobacillus, usually require extended incubation for their detection.     

Direct identification of gram-positive cocci from routine blood cultures by using AccuProbe tests

Rapid and reliable identification of bacteria directly from blood cultures is important in clinical practice to guide appropriate antibiotic therapy. In this study, the performance of the AccuProbe (Gen-Probe, Inc., San Diego, Calif.) in direct identification of Staphylococcus aureus, Streptococcus pneumoniae, enterococci, and group A and B streptococci from positive blood culture bottles was evaluated by using 6-year routine clinical laboratory blood culture material from Paijat-Hame Central Hospital, Lahti, Finland. With the enterococcal and group A and B streptococcal probes, the diagnostic performance of the test was excellent at a cutoff value of 50,000 relative light units (RLU) as recommended by the manufacturer. However, with the S. aureus probe, although the specificity was very high (99.8%), the sensitivity was low (72.4%). To improve the clinical usability of the direct AccuProbe identification, optimal cutoff values for the individual AccuProbe tests were defined by using receiver-operating characteristic analysis. Consequently, cutoff values for S. aureus and S. pneumoniae tests were adjusted to 30,000 RLU and for enterococci and to 55,000 RLU for group A and B streptococci. With these adjustments, the performance of the AccuProbe tests, especially that for S. aureus, was significantly improved.     

Antimicrobial activity of cytotoxic drugs may influence isolation of bacteria and fungi from blood cultures.

The potential antimicrobial activity of cyclophosphamide, vincristine, and adriamycin against Gram positive and negative bacteria and Candida albicans was examined. The time taken for different microbial inocula to turn a simulated blood culture positive in the presence of different concentrations of these drugs was measured. Doxorubicin retarded the growth of Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus sanguis in a concentration dependent manner. Cyclophosphamide and vincristine showed minimal antimicrobial activity. Escherichia coli and Pseudomonas aeruginosa were unaffected by any of the drugs. An inoculum dependent effect was seen with some combinations of microbial inocula and cytotoxic drug concentrations.     

Further studies on the mode of action of Clostridium welchii type-D epsilon toxin.

Intradermal injection of Clostridium welchii type-D epsilon toxin increased the permeability of blood vessels in guinea-pig skin to Evans blue dye by a mechanism not dependent on the release of histamine. The toxin was also found to raise the plasma concentration of cyclic adenosine 3', 5'-monophosphate in mice. It is concluded that epsilon toxin is an enterotoxin capable of causing widespread damage, after binding to receptor sites on the surface of certain cells, through a mechanism mediated by an adenyl cyclase-cAMP system.     

Gas-liquid chromatography in routine processing of blood cultures for detecting anaerobic bacteraemia.

Gas-liquid chromatography was performed on 233 positive blood cultures and findings were compared with culture results. Obligate anaerobic bacteria were recovered from 78 out of 79 blood cultures containing butyric or iso-valeric acids, or both; from 28 out of 69 blood cultures containing succinic acid; and from only one out of 41 blood cultures containing succinic but not butyric or iso-valeric acid. Good correlations (88%) were found for the recovery of anaerobic bacteria and the detection of butyric and/or iso-valeric acids. Detecting volatile fatty acids by gas-liquid chromatography performed on blood cultures at the first signs of growth can therefore provide an early and reliable indication of the presence of anaerobic bacteria.     

Selective medium for isolation of Clostridium botulinum from human feces.

A selective medium, Clostridium botulinum isolation (CBI) agar, was developed for the isolation of C. botulinum from human feces. This medium contains cycloserine (250 microgram/ml), sulfamethoxazole (76 microgram/ml), and trimethoprim (4 microgram/ml) as selective inhibitory agents. Qualitative tests indicated complete recovery of C. botulinum types A, B, F, and G on CBI medium. It was more difficult to recognize type G colonies on the medium because of their lack of lipase activity. Except for a few species of Clostridium, the growth of other obligate anaerobes and of the facultative anaerobes tested on CBI medium was suppressed. Quantitative studies of C. botulinum on the selective medium yielded counts comparable to those obtained on egg yolk agar control plates. Isolation of C. botulinum types A, B, and F from seeded fecal specimens was easily achieved with CBI medium. The use of CBI agar should aid the rapid isolation of C. botulinum from fecal specimens associated with foodborne and infant botulism.     

Selective medium for isolation of Clostridium butyricum from human feces.

The incidence of exfoliative toxin-producing strains of Staphylococcus aureus was studied. Samples from hospitalized patients of all ages and samples from infants less than 6 weeks old were screened; out of 2,632 coagulase-positive S. aureus strains tested, 6.2% synthesized exfoliative toxin. The clinical features could be assessed in 86 patients harboring exfoliative toxin-producing staphylococci. Skin lesions (pustules, blisters, and bullous impetigo) could be observed only when the exfoliative toxin-positive strains were isolated from the skin. Phage nongroup II strains seemed less skin pathogenic than phage group II strains. Outbreaks and sporadic cases were observed.