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.

A five-year multicenter study of the susceptibility of the Bacteroides fragilis group isolates to cephalosporins, cephamins, penicillins, clindamycin, and metronidazole in the United States.

Over 2800 clinical strains of the Bacteroides fragilis group were collected during a 5-year period from ten geographically separate sites and tested for their susceptibility to various antimicrobial agents using a broth microdilution method. Among the cephalosporins, ceftizoxime was the most active (13% resistance) and importantly exhibited relatively equal activity against both B. fragilis species and non-B. fragilis species. Cefotaxime exhibited similar activity with an overall resistance rate of 18%. Both ceftriaxone and cefoperazone were appreciably less active cephalosporins especially against non-B. fragilis species. With regard to cephamycins, cefoxitin (MIC90, 32 micrograms/ml) was more active than cefotetan (MIC90, > or = 256 micrograms/ml) and cefmetazole (MIC90, 64 micrograms/ml). Non-B. fragilis species were highly resistant to cefotetan and cefmetazole. Imipenem was highly active against all strains with the exception of four strains of B. fragilis. Ampicillin-sulbactam, amoxicillin-clavulanate, piperacillin-tazobactam, and cefoperazone-sulbactam were all highly active with resistance rates < 2%. No resistance was detected to metronidazole, whereas 14% of isolates were resistant to clindamycin. When compared with other studies, these findings underscore the wide variability in susceptibility patterns reported nationwide and the need to continue monitoring these patterns to aid in choosing the most active compounds for therapy.

Endocarditis caused by Rochalimaea quintana in a patient infected with human immunodeficiency virus.

Rochalimaea quintana and Rochalimaea henselae are closely related, fastidious, gram-negative rickettsiae. Thus far, the spectrum of human Rochalimaea sp. infections has not included endocarditis. We describe a 50-year-old human immunodeficiency virus-positive man who developed endocarditis caused by R. quintana. DNA relatedness studies, which compared our patient's blood culture isolate with known Rochalimaea species, identified the organism as R. quintana. Our report expands the spectrum of Rochalimaea sp. infections and identifies a new infectious cause of endocarditis.

Characterization of antibiotic-resistant Corynebacterium striatum strains.

Antibiotic-resistant Corynebacterium strains were isolated from 14 Harborview and one Veterans Administration Hospital patients in Seattle during the period 1987-90. These clindamycin-erythromycin resistant strains were shown to hybridize with the ermCd gene, which was cloned from a Corynebacterium diphtheriae plasmid and encodes for a rRNA methylase. Thirteen of these strains also hybridized with the tetM gene probes, and were tetracycline resistant. The ermCd gene could be transferred, by conjugation, while the tetM gene was not transferable.

Helicobacter nemestrinae sp. nov., a spiral bacterium found in the stomach of a pigtailed macaque (Macaca nemestrina)

A new microaerophilic, spirally curved, rod-shaped bacterium was isolated from the gastric mucosa of a pigtailed macaque (Macaca nemestrina). The gram-negative cells of this bacterium are oxidase, catalase, and urease positive and strongly resemble Helicobacter pylori (Campylobacter pylori) cells. Like H. pylori, this organism does not metabolize glucose, does not reduce nitrate or produce indole, does not produce H2S from triple sugar iron agar, does not hydrolyze hippurate or esculin, and does not grow in the presence of 1% glycine, 1.5% salt, or 1% bile. Also like H. pylori, it is resistant to nalidixic acid and susceptible to cephalothin. However, unlike H. pylori, the colorless colonies are flat and have irregular edges. This organism has a unique cellular fatty acid composition, forming a new gas-liquid chromatography group, group K, and a distinctive DNA content (24 mol% guanine plus cytosine). It exhibits less than 10% DNA-DNA homology (as determined by the nylon filter blot method at 65 degrees C) with other members of the genus Helicobacter. Although the levels of DNA relatedness between previously described Helicobacter species and the new organism are low (less than 10%) and the difference in guanine-plus-cytosine content is large (24 versus 36 to 41 mol%), the genus Helicobacter is the only genus in which it is logical to include the organism at this time. We propose that our single strain represents a new species, Helicobacter nemestrinae, and we designate strain T81213-NTB (= ATCC 49396) as the type strain.

Identification of Clostridium difficile in stool specimens by culture-enhanced gas-liquid chromatography.

We have developed a sensitive and specific method for the identification of Clostridium difficile in stool specimens based on the detection of metabolic breakdown products of the organism by gas-liquid chromatography after incubation of stool samples in a selective broth medium containing cefoxitin. Use of this approach to test samples from two different populations of patients at separate medical centers showed this method to be superior to plate cultures or cytotoxin testing alone for both populations. The combined results from the two patient populations showed that 225 of 226 confirmed isolates were identified correctly, resulting in a sensitivity of 99.6% and a specificity of 99.0%. This method eliminates the delay caused by subculturing for tests requiring a pure isolate. The culture phase amplifies even low numbers of C. difficile in fecal samples (due to low in vivo concentrations or delayed transport) and thus increases sensitivity. Other advantages include the ability to detect C. difficile in the mixed flora of the stool and the ability of most clinical laboratories to use this procedure. Given the complexities of the detection of C. difficile toxins and the increasing importance of this organism as a nosocomial agent, culture-based methods remain the preferred approach to screening and routine workup for cases of diarrhea.

Restriction enzyme analysis of plasmid DNA and bacteriophage typing of paired Staphylococcus aureus blood culture isolates.

We compared restriction enzyme analysis of plasmid (REAP) DNA profiling with bacteriophage typing for determination of similarities and differences among 50 pairs of Staphylococcus aureus blood isolates from patients with multiple positive blood cultures. Isolates from 17 pairs did not have detectable plasmids. Isolates from 33 pairs had plasmids classified into 17 distinct REAP DNA profiles. Paired isolates from 31 of these episodes were identical to one another. By phage typing, 35 pairs had strong lytic reactions to a phage(s), 9 pairs lacked strong reactions, and 6 pairs consisted of a strongly reactive isolate and an isolate with no strong reaction to a phage. When consolidated into 11 general phage groups, pairs from 44 of the 50 episodes were in the same general group. REAP DNA profiles were highly reproducible (99%), whereas phage typing was not. REAP DNA profiling is superior to phage typing as a technique for determining similarities and differences among S. aureus blood isolates.

Automated antibiotic susceptibility testing: comparative evaluation of four commercial systems and present state.

Automation of AST has come quite some way and is here to stay. In particular, fully automated, "hands off" instruments have great appeal to laboratories with a limited number of well-trained and experienced clinical microbiology personnel. None of the evaluated instruments is perfect, but then neither are the standard or reference techniques. Overnight incubation has been the yardstick since the early days of in vitro AST. Given the usually shorter therapeutic intervals of 4- to 12-hour dosage schedules, it is quite possible that shorter incubation times for in vitro tests will become more of a standard. Until that time, newer, including automatic, techniques need to be evaluated against the more traditional standard methods. Quality control is critical, and since no systematic approach aside from individual manufacturers' suggestions exists, it should be developed by the NCCLS or similar agencies. Quality control might include standards for the evaluation of such equipment and systems because the development of new technology in this area will continue. Overall, reproducibility and accuracy of the instruments and methods evaluated were quite promising and should encourage well-designed studies of clinical correlation and relevance. The AMS equipment has been in use for routine AST in the clinical laboratories of the Seattle Veterans Administration Medical Center and the University of Washington Hospital. Because of its simplicity and flexibility, the Kirby-Bauer method continues to be an alternate technique for certain important clinical isolates, for instance, blood cultures in both laboratories. Finally, it should be remembered that the most critical function of all such equipment is the reliable detection of resistance.

Campylobacter pylori isolated from the stomach of the monkey, Macaca nemestrina.

Campylobacter pylori was isolated from the gastric mucosa in 6 of 24 pigtailed macaques (Macaca nemestrina) examined by gastric biopsy and culture; 3 isolates were recovered during gastroendoscopy, and 3 were recovered at necropsy. The isolates were morphologically and biochemically similar to the human type strain NCTC 11638, differing only in colony diameter, pigmentation, and rate of growth. Identity of the isolates was confirmed by whole-genomic DNA-DNA hybridization with the type strain. Colonization of the monkey stomachs was associated with hypochlorhydria and histologic features resembling type B chronic gastritis in humans. Host animals exhibited no morbid clinical effects of colonization, although endoscopy revealed inflammation, erythema, and friable tissue in some animals. The discovery of C. pylori occurring spontaneously in M. nemestrina extends the known range of the hosts of the organism and offers the possibility of a natural or experimental model of the infection in monkeys.

Postoperative wound infection surveillance by use of bacterial contamination categories.

A prospective 2-year surveillance of 7129 wounds was conducted on all surgical services of the University Hospital in Seattle to determine the postoperative infection rates by surgical wound category. Rates on all services for clean (0.8%), clean-contaminated (3.4%), contaminated (3.6%), and dirty (9.9%) wounds were recorded and compared to rates reported in the surgical literature. The overall wound infection rate was 1.7%. When the incidence of infection for a specific service in a category was observed to be in excess of a previously reported upper rate, patient charts were critically reviewed to determine if host, pathogen, or technical factors could be implicated in the excessive infection rates. Extending postoperative wound surveillance to include critical chart analysis in these categories provides hospital staff members responsible for infection control the opportunity to organize corrective measures against excessive rates in a broader category of wounds.

Stratified outcome comparison of clindamycin-gentamicin vs chloramphenicol-gentamicin for treatment of intra-abdominal sepsis.

A randomized, prospective trial was conducted of 93 patients with operatively confirmed intra-abdominal sepsis. The study compared clindamycin-gentamicin and chloramphenicol-gentamicin for treatment of carefully stratified patient groups. Malnutrition, age over 65 years, shock, alcoholism, gastrointestinal tract bleeding, steroid administration, diabetes, obesity, and organ malfunction were present with equal frequencies in each group. The duration of antibiotic treatment averaged 8 1/2 days, and the average length of postoperative hospitalization was 29 days. Study antibiotics were changed for bacteriologic reasons in 11 patients taking clindamycin-gentamicin and 12 patients taking chloramphenicol-gentamicin (25% of the total), and two patients in the clindamycin-gentamicin group had a minor adverse reaction. Initial satisfactory clinical responses were obtained in 59 (63%) patients. Twenty-five patients (27%) subsequently developed unsatisfactory courses, but 48 (52%) patients remained well through the 30-day period. Septic-related mortality occurred in 18 (19%) patients, and two (2%) patients had unrelated deaths. There were no significant differences between the study regimens by the outcome criteria evaluated.

Comparison of gram stain, DNA probe, and culture for the identification of species of Mobiluncus in female genital specimens.

The detection of species of Mobiluncus in female genital specimens by DNA probe (with whole-chromosomal bacterial DNA), culture, and gram stain were compared by using specimens obtained from hospital patients, college students, and women attending a sexually transmitted disease clinic. Culture purification and speciation required an average of 37 days to complete, whereas the DNA-probe assay required five days. Gram stain was also rapid but did not allow speciation. There was 100% correlation between species identification by DNA probe and by conventional biochemical tests. Gram stain alone detected 90% of the samples that were positive by any method, whereas culture detected 77%-83% and DNA probe detected 52%-83%.

Instability of antibiotic resistance in a strain of Staphylococcus epidermidis isolated from an outbreak of prosthetic valve endocarditis.

Plasmid profiles, phage typing, antibiograms, and biotyping were used to characterize Staphylococcus epidermidis isolated from multiple cultures of blood of four patients with prosthetic valve endocarditis. Epidemiological evidence implicated a common source for these infections. Of 20 clinically significant isolates, 14 exhibited variations from the prototype pattern of multiple resistance to five antibiotics. All isolates tested appeared to be the same strain by phage typing. Of 18 isolates available for plasmid analysis, 10 contained six plasmids of identical size, whereas eight differed from the prototype profile in the loss of one to three plasmids. Loss of resistance to gentamicin, chloramphenicol, erythromycin, and clindamycin but not to methicillin was associated with the loss of specific plasmids. Because antibiotic resistance in this strain of S. epidermidis was unstable, the use of antibiograms alone was not a reliable means of evaluating the relatedness of these multiple isolates.

Staphylococcus saprophyticus beta-lactamase production and disk diffusion susceptibility testing for three beta-lactam antimicrobial agents.

beta-Lactamase production and MIC determinations for penicillin, methicillin, and cephalothin were assessed for 67 strains of Staphylococcus saprophyticus and correlated with results of disk diffusion susceptibility testing. Fifty-five (82%) of the 67 strains produced beta-lactamase, and 40 (77%) of these beta-lactamase-producing strains were susceptible (zone size, greater than 29 mm) by disk diffusion techniques. Although the range of zone sizes for beta-lactamase producers was broad (26 to 36 mm), all 38 strains with a zone size of less than 31 mm by disk diffusion testing were beta-lactamase producers compared with 17 (59%) of 29 with larger zone sizes (P = 0.0000008). The median penicillin MIC for 12 S. saprophyticus strains was 0.25 micrograms/ml and was not related to beta-lactamase production. Although the methicillin MICs for 15 strains were in the susceptible range (4.0 micrograms/ml), interpretation of disk diffusion testing for oxacillin varied greatly among laboratories using identically prepared media and standardized techniques. Criteria presently used to define susceptibility of Staphylococcus aureus to penicillin and oxacillin by disk diffusion are inappropriate for S. saprophyticus. The clinical significance of the beta-lactamase produced by these strains needs further evaluation.

Nitrocellulose filter blots for species identification of Mobiluncus curtisii and Mobiluncus mulieris.

Seventy strains of Mobiluncus, motile curved anaerobic bacteria associated with bacterial vaginosis, were correctly identified to species level by using bacteria fixed to nitrocellulose and hybridized with 32P-labeled DNA.

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.

Reversal of dementia associated with Whipple's disease by trimethoprim-sulfamethoxazole, drugs that penetrate the blood-brain barrier.

A previously healthy 67-yr-old man presented with progressive dementia over an 11-mo period. Evaluation revealed evidence of malabsorption. Jejunal biopsy established the diagnosis of Whipple's disease. No other etiology for the patient's dementia was uncovered. Treatment with trimethoprim-sulfamethoxazole resulted in rapid elimination of Whipple's bacilli from the jejunum and complete reversal of the patient's dementia over a 6-mo period. Significant levels of trimethoprim and sulfamethoxazole were easily quantitated in the cerebrospinal fluid during therapy. There is increasing recognition of progressive neurologic disease in patients with Whipple's disease who were treated with tetracycline. The reversal of presumed central nervous system disease in this case suggests that drugs that penetrate the blood-brain barrier might be preferable for the initial treatment of Whipple's disease.

An in vitro study of the attachment to vaginal epithelial cells of anaerobic curved rods, Bacteroides bivius and Bacteroides disiens.

An in vitro test system was used to study the influence of different pH levels on the adherence of anaerobic curved rods (ACR) of the long (LCR) and short type (SCR), of Bacteroides bivius and B. disiens, to vaginal epithelial cells (VEC) obtained from healthy women. There was a significant increase in the adherence of ACR (p less than 0.05) and B. bivius (p less than 0.05) at decreasing acidity. LCR adhered significantly better than SCR at pH 7.5 as compared with pH 4.0 (p less than 0.01). 'Clue cells' seen in wet mounts from vaginal discharge of women with bacterial vaginosis were very similar to VEC covered with ACR in in vitro tests.

A serological study of anaerobic curved rods with rabbit hyperimmune antisera.

Anaerobic curved rods (ACR) isolated from vaginal discharge of women with a diagnosis of bacterial vaginosis (BV) were investigated with regard to bacteriologic and serologic characteristics. According to morphologic criteria, long curved rods (LCR) which were sensitive to metronidazole, and short curved rods (SCR) which showed resistance to metronidazole, were recognized. LCR degraded several sugars, whereas SCR degraded only a few carbohydrates. With the use of hyperimmune polyclonal rabbit anti-ACR antibodies, heat-labile and heat-stable antigens were demonstrated in whole cells of both LCR and SCR strains by co-agglutination (CoA). Heat-labile antigens which could be flagellae or other surface components seemed to be responsible for inter-group and inter-strain specificity and did not cross-react between LCR and SCR. Two types of heat-stable antigens, one resistant to 120 degrees C, and one resistant to 100 degrees C but not 120 degrees C, were demonstrated in both LCR and SCR. These heat-stable antigens showed considerable one-way and two-way cross-reactivities within the LCR group, and one-way cross-reactivities were also demonstrated between LCR and SCR strains. These cross-reactivities were also demonstrated by indirect immunofluorescence (IFL) in untreated whole cells. The results show that IFL could be used to identify untreated whole cells and CoA to identify heated bacteria.

Bioactivity of gentamicin in purulent sputum from patients with cystic fibrosis or bronchiectasis: comparison with activity in serum.

Two mechanisms of potential biologic antagonism of gentamicin in purulent sputum from patients with cystic fibrosis or bronchiectasis were studied: reduction of activity by ions and antibiotic binding. Antagonism by ions was assessed by examination of the activity of gentamicin against Pseudomonas aeruginosa in dialysates of serum or sputum in ion-depleted broth. The ionic content of the dialysates increased and reflected differences in the ion content of serum and sputum. Gentamicin had significantly less activity against P aeruginosa in sputum or serum dialysates than in ion-depleted broth alone. When gentamicin was mixed with serum or sputum before dialysis, the level of antipseudomonas activity of the sputum dialysates was significantly lower than that of the serum dialysate; this finding was correlated with greater binding by sputum. Thus, both binding and antagonism by ions evidently reduce the level of bioactivity of gentamicin in serum and in sputum. Purulent sputum, whether from children with cystic fibrosis or adults which bronchiectasis, is more inhibitory than serum; the greater degree of binding, rather than differences in the composition or quantity of cations, explains this difference.