Clonal spread of vancomycin-resistant Enterococcus faecium between patients in three hospitals in two states.

The DNAs of 3* vancomycin-resistant Enterococcus faecium isolates from five hospitals in three states were analyzed by contour-clamped homogeneous electric field electrophoresis and plasmid analysis. There were 22 strain types. One strain type was common to patients in three hospitals in two states. These results suggest the apparent intra- and interhospital spread of vancomycin-resistant E. faecium.

Clinical characteristics of seven cases of diarrhea associated with a novel acid-fast organism in the stool.

In the last 4 years, we have identified an acid-fast, autofluorescent organism in the stool of seven patients with diarrhea. The organism was identified as a cyanobacterium-like organism by the Centers for Disease Control (Atlanta) and as a coccidian by researchers in Peru and at the University of Arizona (Tucson). We present reports on the seven cases and a review of the literature. Three patients were known to be seropositive for the human immunodeficiency virus (HIV). All complained of watery diarrhea that had begun 3 days to 8 months before presentation. Colonoscopy in two patients showed a normal mucosal pattern; a biopsy for one of these patients showed chronic nonspecific inflammation of the colon. Examination of stool for ova and parasites revealed multiple oval and round nonrefractile organisms with well-defined walls that resembled large cryptosporidia; they measured 8-9 microns in diameter. These organisms did not stain by Giemsa or gram methods but were acid-fast by Kinyoun carbolfuchsin and Ziehl-Neelsen stains. The organisms fluoresced as blue under ultraviolet light. In the immunocompetent patients, diarrhea lasted an average of 19 days and resolved spontaneously. Diarrhea persisted in the HIV-seropositive patients. The clinical course and organism resembled those reported for travelers and HIV-seropositive patients. This organism may represent a newly identified cause of watery diarrhea in humans.

Molecular typing of ampicillin-resistant, non-beta-lactamase-producing Enterococcus faecium isolates from diverse geographic areas.

Molecular typing methods were compared by using 66 ampicillin-resistant, non-beta-lactamase-producing Enterococcus faecium clinical isolates from diverse geographic areas. Whole-plasmid analysis, restriction enzyme analysis of plasmid DNA with EcoRI and HindIII, and contour-clamped homogeneous electric field electrophoresis with digestion by SmaI and ApaI were performed on all isolates. Whole-plasmid analysis identified 47 different groups. Restriction enzyme analysis of plasmid DNA identified 50 groups when EcoRI was used and 51 groups when HindIII was used. Results with EcoRI and HindIII differed in 9 of 66 isolates. Grouping results with whole-plasmid analysis differed from results of restriction enzyme analysis of plasmid DNA (combining EcoRI and HindIII) in 20 of 66 isolates. Contour-clamped homogeneous electric field electrophoresis identified 46 groups when SmaI was used and 44 groups when ApaI was used. Results with SmaI and ApaI differed in 3 of 66 isolates. Grouping results with contour-clamped homogeneous electric field electrophoresis (combining SmaI and ApaI) differed from results of restriction enzyme analysis of plasmid DNA (combining EcoRI and HindIII) in 17 of 66 isolates. The combined use of whole-plasmid analysis, restriction enzyme analysis of plasmid DNA with two enzymes, and contour-clamped homogeneous electric field electrophoresis with two restriction enzymes should be considered when E. faecium is typed for epidemiologic investigation.

The emergency management of moray eel bites.

As human encounters with hazardous marine life increase, emergency physicians are more frequently confronted with the management of resultant injuries. We present three cases involving hand injuries inflected by moray eels. Each was managed with local wound care and subsequent outpatient treatment with either oral ciprofloxacin or cefuroxime. One patient had mild residual hand dysfunction, and no patient developed wound infection. To better assess the bacteriology of such injuries, oral cultures were taken from captive moray eels and surrounding aquarium water. Culture and sensitivity analyses showed Vibrio and Pseudomonas to be the predominant species, both sensitive to ciprofloxacin, cefuroxime, tetracycline, and trimethoprim-sulfamethoxazole. We conclude that moray eel bites can be managed successfully with aggressive, local wound care and antibiotic coverage that targets Vibrio and Pseudomonas species.

Aeromonas hydrophila infection associated with the use of medicinal leeches.

The use of medicinal leeches (Hiruda medicinalis) is becoming more common after plastic surgery to control venous congestion of skin grafts. We describe a patient with Aeromonas hydrophila infection whose graft was treated with medicinal leeches. The infection required systemic antibiotic therapy. A. hydrophila is the predominant bacterial flora in the gut of the leech, where it plays an essential role for the animal in the digestion of blood. The potential for A. hydrophila wound infection, and appropriate antibiotic prophylaxis of the leech or patient, should be considered when medicinal leeches are used.

Identification of Neisseria gonorrhoeae with the ORTHOProbe DNA probe test.

We evaluated and compared the ORTHOProbe DNA hybridization test (Ortho Diagnostic Systems, Inc., Raritan, NJ) with conventional carbohydrate fermentation and commercial rapid tests for identifying gonococci (GC). The ORTHOProbe system employs a biotinylated DNA probe that hybridizes specifically with chromosomal target sequences of GC. Hybridization is detectable visually following the addition of a streptavidin-peroxidase conjugate in a rapid 10-min. assay. A total of 200 clinical isolates resembling Neisseria (gram-negative, oxidase-positive diplococci) cultured from various sites were tested. The ORTHOProbe test reacted strongly with all GC (94) and not with nongonococcal organisms (106), yielding a sensitivity and specificity of 100%. The ORTHOProbe system provides a convenient and reliable test for identifying Neisseria gonorrhoeae.

Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes.

DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA probes for identification of clinically important Bacteroides species.

Conventional procedures for identifying gram-negative anaerobes such as Bacteroides spp. are cumbersome and time-consuming. A simpler approach would be to use DNA probes to identify these organisms. Since many different species of gram-negative anaerobes are isolated from clinical specimens, the most useful DNA probes would be probes that identify a few clinically significant groups of anaerobes. To obtain such probes, we cloned HindIII-digested chromosomal DNA from various Bacteroides species and then screened these probes, labeled with 32P by nick translation, for hybridization to DNA from various species of Bacteroides, Fusobacterium, and other gram-negative bacteria. We identified three DNA probes (pBFII-4, pBFII-5, and pBFII-6) that hybridized specifically to DNA from Bacteroides fragilis, the species that accounts for about half of all isolates from clinical specimens containing anaerobes. We identified one DNA probe (pBE-3) that hybridized to all members of the B. fragilis group, a group of species that resemble B. fragilis with respect to fermentation pattern and antibiotic resistances. We also identified one probe (pBO-21) that hybridized to DNA from all Bacteroides sp. strains as well as to DNA from strains of Fusobacterium necrophorum and Fusobacterium nucleatum. pBO-21, but not pBE-3, cross-hybridized with a cloned Bacteroides sp. 16S rRNA gene. The limit of detection for these probes was 10(6) bacteria. The probes could detect B. fragilis in blood culture medium and in mixed cultures with other gram-negative bacteria. Attempts to use biotin-labeled DNA probes instead of 32P-labeled probes were not successful because the Bacteroides sp. extracts contained material that bound the streptoavidin-peroxidase detection reagent.

Use of a species-specific DNA hybridization probe for enumerating Bacteroides vulgatus in human feces.

pBV-1, a recombinant plasmid that contains a chromosomal DNA fragment from Bacteroides vulgatus, hybridized to DNA from B. vulgatus but not to DNA from other colonic Bacteroides species. This plasmid was used as a DNA probe to detect and enumerate B. vulgatus in pure culture, in mixed cultures, and in a bacterial fraction from human feces. Bacteria in a pure or mixed culture were lysed by heating the culture in NaOH. The DNA in the disrupted cell suspension was then trapped on nitrocellulose paper by vacuum filtration. If fecal samples were used instead of pure or mixed cultures, it was first necessary to partially purify the DNA by low-speed centrifugation (2,000 X g) and phenol-chloroform extraction before filtering. When 32P-labeled pBV-1 was incubated with filters containg B. vulgatus DNA, the amount of radioactivity that bound to the filters was proportional to the number of B. vulgatus filtered as long as the filtering capacity of the nitrocellulose was not exceeded. Using this procedure, we obtained a value for the concentration of B. vulgatus in human feces (2 X 10(10) to 3 X 10(10) per g of dry weight) that is similar to values obtained by other investigators using conventional bacteriological techniques (3 X 10(10) to 6 X 10(10) per g of dry weight). The advantage of the DNA hybridization method over conventional techniques is that it is not necessary to isolate pure cultures of bacteria from complex specimens such as feces. Furthermore, our method bypasses the cumbersome set of biochemical tests normally used to identify anaerobic bacteria. The major limitation of our method is its sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Digestion of proteoglycan by Bacteroides thetaiotaomicron.

It has been shown previously that Bacteroides thetaiotaomicron, a human colonic anaerobe, can utilize the tissue mucopolysaccharide chondroitin sulfate as a source of carbon and energy and that the enzymes involved in this utilization are all cell associated (A. A. Salyers and M. B. O'Brien, J. Bacteriol. 143:772-780, 1980). Since chondroitin sulfate does not generally occur in isolated form in tissue, but rather is bound covalently in proteoglycan, we investigated the extent to which chondroitin sulfate which is bound in such a sterically hindered complex can be utilized by intact bacteria. Intact cells of B. thetaiotaomicron were able to digest chondroitin sulfate in proteoglycan, although at a slightly slower rate than free chondroitin sulfate. Prior digestion of proteoglycan with trypsin to produce small fragments of protein with several chondroitin sulfate chains attached did not increase the rate at which the bound chondroitin sulfate was digested. Accordingly, the slower rate of digestion was probably due to attachment of chondroitin sulfate chains to the protein backbone rather than to steric hindrance by other components of the proteoglycan. When proteoglycan which had been incubated with intact bacteria was treated with sodium borohydride to release the undigested fragments of chondroitin sulfate from the protein backbone, the size and composition of the fragments indicated that intact bacteria were able to digest all but three monosaccharides of the chondroitin sulfate chains. Thus, despite steric hindrance due to attachment of the chondroitin sulfate chains to the protein backbone, digestion of bound chondroitin sulfate by intact bacteria was nearly complete.