Comparison of Escherichia coli O157:H7 antigen detection in stool and broth cultures to that in sorbitol-MacConkey agar stool cultures.

We evaluated the Meridian IC-STAT direct fecal and broth culture antigen detection methods with samples from children infected with Escherichia coli O157:H7 and correlated the antigen detection results with the culture results. Stools of 16 children who had recently had stool cultures positive for this pathogen (population A) and 102 children with diarrhea of unknown cause (population B) were tested with the IC-STAT device (direct testing). Fecal broth cultures were also tested with this device (broth testing). The results were correlated to a standard of the combined yield from direct culture of stools on sorbitol-MacConkey (SMAC) agar and culture of broth on SMAC agar. Eleven (69%) of the population A stool specimens yielded E. coli O157:H7 when plated directly on SMAC agar. Two more specimens yielded this pathogen when the broth culture was similarly plated. Of these 13 stool specimens, 8 and 13 were positive by direct and broth testing (respective sensitivities, 62 and 100%). Compared to the sensitivity of a simultaneously performed SMAC agar culture, the sensitivity of direct testing was 73%. Three (3%) of the population B stool specimens contained E. coli O157:H7 on SMAC agar culture; one and three of these stool specimens were positive by direct and broth testing, respectively. The direct and broth IC-STAT tests were 100% specific with samples from children from population B. Direct IC-STAT testing of stools is rapid, easily performed, and specific but is insufficiently sensitive to exclude the possibility of infection with E. coli O157:H7. Performing the IC-STAT test with a broth culture increases its sensitivity. However, attempts to recover E. coli O157:H7 by culture should not be abandoned but, rather, should be increased when the IC-STAT test result is positive.

Activities of tobramycin and six other antibiotics against Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

The in vitro activity of tobramycin was compared with those of six other antimicrobial agents against 1,240 Pseudomonas aeruginosa isolates collected from 508 patients with cystic fibrosis during pretreatment visits as part of the phase III clinical trials of tobramycin solution for inhalation. The tobramycin MIC at which 50% of isolates are inhibited (MIC(50)) and MIC(90) were 1 and 8 microg/ml, respectively. Tobramycin was the most active drug tested and also showed good activity against isolates resistant to multiple antibiotics. The isolates were less frequently resistant to tobramycin (5.4%) than to ceftazidime (11.1%), aztreonam (11.9%), amikacin (13.1%), ticarcillin (16.7%), gentamicin (19.3%), or ciprofloxacin (20.7%). For all antibiotics tested, nonmucoid isolates were more resistant than mucoid isolates. Of 56 isolates for which the tobramycin MIC was > or = 16 microg/ml and that were investigated for resistance mechanisms, only 7 (12.5%) were shown to possess known aminoglycoside-modifying enzymes; the remaining were presumably resistant by an incompletely understood mechanism often referred to as "impermeability."

Microbiology of sputum from patients at cystic fibrosis centers in the United States.

During a phase III national collaborative study of aerosolized tobramycin (1 July 1995 through 30 September 1996), the microbiology of specimens from 595 patients at 69 cystic fibrosis (CF) centers was examined. Samples from three screening visits were processed in a single laboratory by means of standardized techniques for identification and susceptibility testing. From 1,753 pretreatment specimens, 5,128 pathogens were isolated (average, 2.9/specimen). Of the 3,936 Pseudomonas aeruginosa isolates, 56.7% were mucoid. The specimens of 125 patients (21.0%) yielded tobramycin-resistant P. aeruginosa (213 isolates); 61 (10.3%), Stenotrophomonas maltophilia; and 52 (8.7%), Alcaligenes xylosoxidans. Isolation of Burkholderia cepacia was an exclusion criterion. Only visit 3 sputum samples were cultured for gram-positive organisms and fungi (n = 465 patients); samples from 201 patients (43.2%) yielded Staphylococcus aureus (18.8% of isolates were oxacillin-resistant), and those from 114 (24.5%) yielded an Aspergillus species. Compared with the Cystic Fibrosis Foundation Patient Registry, the current study identified many more patients colonized with S. maltophilia, A. xylosoxidans, Aspergillus species, and oxacillin-resistant S. aureus, suggesting the utility of standardized processing of CF specimens.

Genetic and phenotypic analysis of Escherichia coli with enteropathogenic characteristics isolated from Seattle children.

Coliform colonies from children whose stools were submitted for microbiologic analysis were studied prospectively to determine the frequency of shedding of enteropathogenic Escherichia coli (EPEC). In total, 2225 isolates from 445 patients were probed with eaeA (encoding intimin) and the EAF (EPEC adherence factor) probe, and adherence and actin-aggregating phenotypes were determined. Twenty-five patients (5.6%) shed non-O157:H7 eaeA+ EAF- E. coli. Of these 25 patients, isolates from 5 produced Shiga toxins and from 3 possessed bfpA (encoding the bundle-forming pilus) sequences. Non-O157:H7 eaeA+ E. coli from 21 (84%) of 25 patients adhered locally to and aggregated actin in HeLa cells. Four patients shed nonadherent EAF+ eaeA- E. coli. Non-O157:H7 eaeA+ and EAF- isolates belonged to diverse electrophoretic types and classical and nonclassical enteropathogenic serotypes. EPEC are relatively common in stools submitted for analysis in this North American pediatric hospital. Their etiologic role in childhood diarrhea warrants elucidation.

Use of the fluorochrome calcofluor white in the screening of stool specimens for spores of microsporidia.

Microsporidia are obligate intracellular protozoal pathogens associated with chronic diarrhea in individuals infected with HIV. Direct detection methods for microsporidial spores in stool include chromotrope-based, fluorochrome, and immunofluorescent stains. The authors compared the ability to detect microsporidial spores in 168 stool specimens using two stains: a chromotrope-based modified trichrome stain and a fluorochrome stain, calcofluor white (Cellufluor, Polysciences, Warrington, PA). In addition to being faster and easier to perform, the calcofluor white stain was found to be more sensitive than the chromotrope-based stain, as 6 of 24 specimens positive by calcofluor white were negative by the chromotrope-based stain on initial smear evaluation. Repeat examination confirmed these six as being positive. To evaluate the specificity of the calcofluor white stain, 20 formalin-fixed stool specimens (5 positive and 15 negative for microsporidial spores) were evaluated in blinded fashion by two affiliated clinical laboratories using their own formulations of calcofluor white. A single discrepant result (falsely positive) was reported from one laboratory. The use of the calcofluor white stain is recommended as a simple and highly sensitive screening procedure for the detection of microsporidial spores in stool specimens.

Characteristics of antibiotic-resistant Escherichia coli O157:H7 in Washington State, 1984-1991.

The resistance of Escherichia coli O157:H7 to amoxicillin/clavulanic acid, ampicillin, ceftazidime, ceftriaxone, cefuroxime, cephalothin, chloramphenicol, ciprofloxacin, gentamicin, streptomycin, sulfisoxazole, tetracycline, ticarcillin, tobramycin, and trimethoprim-sulfamethoxazole was examined, and resistant strains were characterized. All 56 isolates collected between 1984 and 1987 were susceptible to all antibiotics tested; 13 (7.4%) of 176 strains isolated between 1989 and 1991 were resistant to streptomycin, sulfisoxazole, and tetracycline. lambda-restriction fragment length polymorphism analysis suggested that the 13 resistant strains belonged to nine different clones. The emerging resistance of E. coli O157:H7 to antibiotics could portend an increased prevalence of this pathogen in food animals that receive antibiotics. Antimicrobial resistance of E. coli O157:H7 could be useful as a rapid epidemiologic marker and as a way to select this pathogen from suspected vehicles of transmission, but this resistance could also complicate therapeutic trials with sulfa-containing antibiotics.

Shiga-like toxin-producing Escherichia coli in Seattle children: a prospective study.

BACKGROUND: The frequency with which stools contain Shiga-like toxin producing Escherichia coli not belonging to serotype O157:H7 is unknown in the United States. The aim of this study was to determine the frequency with which these E. coli are present in stools from children from Seattle submitted for bacteriologic analysis. METHODS: 2225 coliform colonies from 445 stools submitted for bacterial culture from Seattle children were probed with the structural genes of Shiga-like toxins I and II in a 1-year prospective study. The adherence and actin aggregating characteristics of these E. coli were subsequently determined. RESULTS: Five (1.1%) patients had non-O157:H7 Shiga-like toxin producing E. coli, a rate of isolation higher than Shigella or Yersinia (0.2% each) but lower than Campylobacter (2.5%), E. coli O157:H7 (2.9%), or Salmonella (3.4%). Only one of the five patients had bloody diarrhea. None developed hemolytic uremic syndrome. All strains adhered in a localized pattern to, and induced actin aggregation in, HeLa cells, and produced a toxin that was lethal to Vero cells. CONCLUSIONS: Non-O157:H7 Shiga-like toxin producing E. coli are present in stools submitted for bacterial culture in a North American childhood population. Their role in childhood diarrhea warrants better definition.

Bacteriology of acute otitis media: a new perspective.

Pathogenic bacteria were isolated from 90% of patients with acute otitis media. This higher-than-expected rate of positive cultures was probably related to the meticulous bacteriologic techniques used.

Escherichia coli O157:H7 and the hemolytic uremic syndrome: importance of early cultures in establishing the etiology.

Fifty-two patients were studied prospectively to determine the etiology of postdiarrheal hemolytic uremic syndrome (HUS). Escherichia coli O157:H7 was isolated from 33 patients (63.4%). If stool obtained within 2 days of the onset of diarrhea was cultured for E. coli O157:H7, the recovery rate was 100%. This rate decreased to 91.7% and 33.3% if stool was cultured for this pathogen 3-6 or greater than or equal to 7 days, respectively, after diarrhea began. The culture-positive group was more likely to have had bloody diarrhea and fecal leukocytes and to have received transfusions than the culture-negative group but was otherwise similar in clinical characteristics. E. coli O157:H7 is the predominant pathogen associated with HUS in western Washington. Recovery of this pathogen is highly dependent on obtaining stool cultures within 6 days of onset of diarrhea.

Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cells.

A fimbrial adhesin, designated F1845, was found to be responsible for the diffuse HEp-2 cell adherence of a diarrheal Escherichia coli isolate. The genetic determinant of F1845 was cloned, and the order of the genes necessary for production of F1845 was determined by maxicell analysis. Five polypeptides with apparent sizes of 10, 95, 27, 15.5, and 14.3 kilodaltons (kDa) were found to be encoded in that order by the F1845 determinant. The nucleotide sequence of the 14.3-kDa subunit gene was determined and found to share extensive homology in its signal sequence with the gene encoding the structural subunit of the AFA-1 hemagglutinin of a uropathogenic E. coli strain (A. Labigne-Roussel, M.A. Schmidt, W. Walz, and S. Falkow, J. Bacteriol. 162:1285-1292, 1985) but not in the region encoding the mature protein. Southern blot hybridizations indicated that the F1845 determinants are of chromosomal origin. Hybridization studies using a probe from the region encoding the 95-kDa polypeptide indicated that related sequences may be plasmid associated in some strains and chromosomal in others. Additional hybridization studies of E. coli isolates possessing sequence homology to the F1845 determinant suggest that the sequences in the 5' region of the F1845 structural subunit gene are more highly conserved than sequences in the 3' region.

Escherichia coli O157:H7 as the predominant pathogen associated with the hemolytic uremic syndrome: a prospective study in the Pacific Northwest.

During a 12-month period, 14 patients with the hemolytic uremic syndrome were identified in a prospective study of enteric pathogens associated with this disorder. Of the 12 patients with a diarrheal illness preceding the onset of hemolytic uremic syndrome, fecal Escherichia coli O157:H7 was detected in seven (58%), all of whom had bloody diarrhea. Half of the siblings of these patients had concurrent nonbloody diarrhea. No source for infection with this organism was identified. Enteric infection with E coli O157:H7 occurs in the majority of cases of hemolytic uremic syndrome following diarrheal illness in the Pacific Northwest and may represent a previously overlooked cause of hemolytic uremic syndrome in other locales. Evaluation of all cases of hemolytic uremic syndrome for enteric pathogens should routinely include cultures for E coli O157:H7 until results of additional studies clarify the distribution of agents associated with hemolytic uremic syndrome in different geographic regions. These findings may provide new opportunities for the design of therapeutic and preventive strategies in this disorder.

Diarrhea associated with adherent enteropathogenic Escherichia coli in an infant and toddler center, Seattle, Washington.

During November 1983, the Seattle-King County Department of Public Health investigated an outbreak of diarrhea associated with enteropathogenic Escherichia coli, serogroup 0111:K58, in an infant and toddler day-care center. Of the 25 children in the center, ranging in age from 4 to 30 months (median age 11 months), diarrhea occurred in 14 characterized by watery, greenish stools. The median duration of diarrhea was 12 days. Two of the ill children were hospitalized because of severe dehydration. Stool cultures from the children diagnosed initially did not yield the common bacterial pathogens, parasites, or rotavirus. Stool cultures from 11 of 14 ill children and two of 11 well children (P less than .005), however, yielded an E coli serogroup, 0111:K58, which was not invasive or toxigenic by standard tests. The source of the organism was not identified. Although this organism has been recognized as a cause of diarrhea in newborn nurseries, this is the first published report of a documented outbreak of enteropathogenic E coli-induced diarrhea in a day-care center in the United States.

The use of acridine orange as a rapid method for the quantitation of bacteremia in laboratory animals.

A rapid technique has been developed to quantitate the degree of bacteremia in laboratory animals. Direct staining of blood smears with acridine orange and enumeration using fluorescent microscopy allowed quantitation of Haemophilus influenzae in blood at densities from 10(5) to 10(8) cfu/ml. This technique will facilitate the accuracy with which therapeutic trials are conducted in laboratory models of infection.

Rapid antimicrobial susceptibility testing of isolates from blood cultures by direct inoculation and early reading of disk diffusion tests.

Disk diffusion tests, inoculated directly from positive blood cultures, were evaluated for accuracy of reading zone diameters after 4- and 6-h and overnight incubation. In comparisons with results from standard disk diffusion tests, the 4-h results were in agreement for 83% of tests with gram-positive organisms and 64% of tests with gram-negative organisms. When minor discrepancies were ignored, the 4-h readings were in agreement for 98% of the tests with gram-positive organisms and 95% of the tests with gram-negative organisms. After 6 h of incubation, 91% of the tests with gram-positive organisms and 86% of the tests with gram-negative organisms agreed with standard results. The agreement was 99% for tests with both gram-positive and gram-negative organisms when minor discrepancies were excluded. Very major discrepancies occurred in two tests (0.1%) with gram-positive organisms and were not observed in tests with gram-negative organisms. The frequencies of major discrepancies were 3.5% after 4 h, 0.6% after 6 h, and 0.7% after overnight incubation. Ampicillin and cephalothin tests with Escherichia coli and Klebsiella spp. accounted for 81% of the major discrepancies in tests with gram-negative organisms. Oxacillin tests accounted for more than half of the major discrepancies in tests with staphylococci. The results of this study, which did not include the newer antibiotics, indicate that direct susceptibility tests from blood cultures read after 6 h of incubation are more reliable than 4-h results and produce less than 1% major errors in comparisons with standard susceptibility tests.