First comprehensive evaluation of the M.I.C. evaluator device compared to Etest and CLSI reference dilution methods for antimicrobial susceptibility testing of clinical strains of anaerobes and other fastidious bacterial species.

The new M.I.C. Evaluator strip uses test methodology and the recording of results that are similar to those of Etest. For this first assessment, 102 clinical strains of anaerobic bacteria from 12 genera and 155 strains from 7 genera and 8 species of fastidious bacteria were tested by M.I.C. Evaluator, Etest, and agar dilution or broth microdilution as a reference standard. Ampicillin, amoxicillin, amoxicillin-clavulanate, cefotaxime, ciprofloxacin, erythromycin, imipenem, levofloxacin, metronidazole, penicillin, and tetracycline were tested depending on the species. Agar dilution for anaerobes was performed according to CLSI document M11-A7. For the fastidious bacteria, CLSI document M45-A2 was followed. For the anaerobes, essential and categorical agreement between M.I.C. Evaluator and Etest was >90%. Compared to agar dilution, essential agreement was low for both strip tests, and many very major errors were observed for metronidazole (13 to 14%) and penicillin (8 to 9%) with isolates from the Bacteroides fragilis group and Clostridium species. For fastidious species, essential agreements for M.I.C. Evaluator and Etest plus or minus one doubling dilution were >95%. Compared to broth microdilution, essential agreements were low (40 to 90%) plus or minus one dilution and were >90% plus or minus two dilutions, with high overall category agreement (CA). Major and minor errors were within established parameters for all strains tested. The M.I.C. Evaluator strips were equivalent to Etest for anaerobes and fastidious species. These observations require further investigation to determine which methods provide the most accurate MIC for clinical utility. The further evaluation of additional M.I.C. Evaluator agents will be performed as they become available.

First comprehensive evaluation of the M.I.C. evaluator device compared to Etest and CLSI broth microdilution for MIC testing of aerobic Gram-positive and Gram-negative bacterial species.

The M.I.C. Evaluator strip (Thermo Fisher Scientific, Basingstoke, United Kingdom) uses a methodology similar to that of Etest. In this first assessment of the M.I.C. Evaluator device, 409 strains of aerobic Gram-positive bacteria (staphylococci, streptococci, and enterococci) and 325 strains of Enterobacteriaceae, Pseudomonas species, and Acinetobacter species were tested by M.I.C. Evaluator strip, Etest, and broth microdilution as a reference standard. The Gram-positive bacteria included staphylococci (methicillin-resistant Staphylococcus aureus, methicillin-susceptible S. aureus, and coagulase-negative staphylococci), Streptococcus pneumoniae, beta-hemolytic streptococci and viridians group strains, vancomycin-resistant enterococci, and other enterococci. The Gram-negative bacteria included 250 strains of 60 Enterobacteriaceae species plus 50 Pseudomonas and 25 Acinetobacter species. A total of 14 antimicrobial agents (depending on the species) were included. The same methodology and reading format were used for M.I.C. Evaluator strips and Etest. Broth microdilution methodology was performed according to CLSI document M07-A8. For the clinical strains, >95% of results were plus or minus one doubling dilution for all species. There were fewer than 5% minor errors, fewer than 3% major errors, and fewer than 1% very major errors. M.I.C. Evaluator strips and Etest often reported higher MICs than the reference broth microdilution method. The M.I.C. Evaluator strips provided results comparable to those of the predicate Etest device and are of value for the accurate testing of MICs for these important pathogens.

Precision and accuracy of fluconazole susceptibility testing by broth microdilution, Etest, and disk diffusion methods.

Interpretive agreements among the results of fluconazole broth microdilution tests, Etests, and disk diffusion tests were documented by evaluating 495 Candida spp. Microdilution reference test results were in agreement with 96% of the Etest results; most discrepancies were minor differences. Fluconazole resistance of Candida krusei strains often required a full 48 h of incubation in order to be observed by the standard method. For the disk diffusion tests that were performed on Mueller-Hinton agar with glucose and methylene blue, 97% of results were in agreement with those of the reference test, especially when zones of inhibition were measured after the first 24 h of incubation. Some Candida glabrata isolates failed to grow satisfactorily until a full 48 h of incubation was completed. Precision was determined by testing 50 selected isolates in triplicate in each of three laboratories. The reproducibility of results of disk diffusion tests was comparable to that of the reference method. With all procedures, determination of test results was particularly challenging with some strains, and new methods are needed in order to improve endpoint definition.

Detection of anthrax spores in endemic regions of northern Canada.

AIMS: To determine the level of anthrax spore contamination in endemic regions of northern Canada between outbreaks. METHODS AND RESULTS: Bacterial endospores were extracted from specimens via flotation and cultured on selective PLET medium. Of 588 environmental specimens collected, 11 (1.9%) contained viable anthrax spores. CONCLUSION: High environmental concentrations of anthrax spores in northern Canada appear limited to scavenger faeces and anthrax carcass sites. Burial and cremation appear equally effective at removing anthrax spores from the immediate environment, though cremation may be improved by re-burning cremation sites containing unburned animal hair. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes an effective anthrax spore detection system. It provides the first bacteriological evidence that mammalian scavengers can disseminate anthrax spores in northern Canada, and its results may be compared with future environmental studies of untreated anthrax carcass sites to help improve government response plans.

Evaluation of E test, disk diffusion and broth microdilution to establish tentative quality control limits and review susceptibility breakpoints for two aerobic actinomycetes.

A single laboratory study was carried out to compare E Test with broth microdilution and disk diffusion to establish tentative quality control ranges for Nocardia asteroides ATCC 19247 and Rhodococcus equi ATCC 6939 against a panel of eight antimicrobial agents. Reproducibility testing was performed on 12 consecutive days to establish tentative quality control ranges. A total of 36 clinical strains of the Nocardia asteroides complex and 5 Rhodococcus strains were used in the study. Both candidate control strains and clinical strains grew well on cation-adjusted Mueller-Hinton agar. Adequate growth occurred at 48 to 72 h for the Nocardia isolates and 24 to 48 h for Rhodococcus. A standardized primary inoculum of 5 x 10(4) CFU/mL was used for performance of E Test and disk diffusion for the Nocardia isolates. Tentative population-based error rates were calculated using current breakpoints for Enterobacteriaceae for E Test compared with disk diffusion for the 36 clinical strains of Nocardia species. Significant very major error rates were observed for imipenem (22%) and minor error rates varied from 2.7% to 50%. These methods require more extensive validation before definitive breakpoint criteria can be established.

Evaluation of spore extraction and purification methods for selective recovery of viable Bacillus anthracis spores.

AIMS: To investigate methods of improving anthrax spore detection with PLET. METHODS AND RESULTS: Comparisons were made of PLET and blood-supplemented PLET to recover and distinguish spores of a variety of Bacillus species. Heat and ethanol purification of spores, and spore extraction from soil with water and high specific gravity sucrose plus non-ionic detergent, were also carried out. CONCLUSION: PLET was more selective and suitable than blood-supplemented PLET for detection of anthrax spores in the environmental specimens. However, PLET is not an optimal spore recovery medium. Purification of spores with ethanol was as effective as heat purification. High specific gravity sucrose plus detergent extraction solutions may be more sensitive than extraction with water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights shortcomings with the standard PLET isolation of anthrax spores and describes ways in which the procedure may be improved.

Novel fluorescent broth microdilution method for fluconazole susceptibility testing of Candida albicans.

A comparative evaluation of the reference National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method with a novel fluorescent carboxyfluorescein diacetate (CFDA)-modified microdilution method for the susceptibility testing of fluconazole was conducted with 68 Candida strains, including 53 Candida albicans, 5 Candida tropicalis, 5 Candida glabrata, and 5 Candida parapsilosis strains. We found trailing endpoints and discordant fluconazole MICs of < 8 microg/ml at 24 h and of > or =64 microg/ml at 48 h for 12 of the C. albicans strains. These strains satisfy the definition of the low-high MIC phenotype. All 12 low-high phenotype strains were correctly shown to be susceptible at 48 h with the CFDA-modified microdilution method. For the 41 non-low-high phenotype C. albicans strains, the CFDA-modified microdilution method yielded 97.6% (40 of 41 strains) agreement within +/-1 dilution at 24 h compared with the reference method and 92.7% (38 of 41 strains) agreement within +/-1 dilution at 48 h compared with the reference method. The five strains each from C. tropicalis, C. glabrata, and C. parapsilosis that were tested showed 100% agreement within +/-2 dilutions for the two methods being evaluated.

Quality control limits for broth microdilution susceptibility tests of ten antifungal agents.

Broth microdilution susceptibility tests of Candida species have now been standardized by the National Committee for Clinical Laboratory Standards (NCCLS). An eight-laboratory collaborative study was carried out in order to document reproducibility of tests of Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 by the NCCLS method. Replicate broth microdilution tests were used to define control limits for 24- and 48-h MICs of amphotericin B, flucytosine, fluconazole, voriconazole, ketoconazole, itraconazole, caspofungin (MK 0991), ravuconazole (BMS 207147), posaconazole (SCH 56592), and LY 303366.

Antibiotic therapy attenuates colitis in interleukin 10 gene-deficient mice.

BACKGROUND & AIMS: Interleukin (IL)-10 gene-deficient mice, raised under germfree conditions, do not develop colitis, implying a role for bacteria. This study mapped the appearance of luminal colonic bacteria and, using antibiotic treatment, determined their association with colitis in IL-10 gene-deficient mice. METHODS: Mice were treated with ciprofloxacin or with neomycin and metronidazole. The intestine was harvested for histological scoring and bacterial assessment. RESULTS: At 2 weeks of age, before the development of colitis, IL-10 gene-deficient mice demonstrated an earlier appearance of Streptococcus and Clostridium sp., and had a greater proportion (P < 0.01) of bacteria adherent to the colonic mucosa. This pattern of increased adherent bacteria persisted for the 12 weeks of study. Treatment of mice before the onset of colonic inflammation, with either antibiotic regime, reduced mucosal adherent bacteria and prevented colitis (P < 0.01). In contrast, treatment of established colitis with neomycin and metronidazole did not reduce adherent bacterial levels, yet was more efficacious (P < 0.05) in treating established colitis than ciprofloxacin, which did reduce adherent colonic bacteria. CONCLUSIONS: In the IL-10 gene-deficient mouse model, the appearance and number of mucosal adherent colonic bacteria are altered before the onset of colitis. Antibiotics both prevent and treat the colitis through correction of this primary bacterial alteration.

Rapid identification of clinical yeast isolates using the colorimetric AUXACOLOR system.

The AUXACOLOR colorimetric system (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) for the identification of clinical yeast isolates, was compared in its identification of 100 yeast strains to conventional identification methods. Of the 94 correctly identified isolates, 47% (n = 44) were identified by 24 h, and 100% (n = 94) were identified by 48 h. AUXACOLOR is a simple, rapid and accurate method for the identification of yeast pathogens.

Assessment of the effect of amphotericin B on the vitality of Candida albicans.

The processes involved in cell death are complex, and individual techniques measure specific fractions of the total population. The interaction of Candida albicans with amphotericin B was measured with fluorescent probes with different cellular affinities. These were used to provide qualitative and quantitative information of physiological parameters which contribute to fungal cell viability. SYBR Green I and 5,(6)-carboxyfluorescein were used to assess membrane integrity, and bis-(1,3-dibutylbarbituric acid)trimethine oxonol and 3,3-dihexyloxacarbocyanine iodide were used to evaluate alterations in membrane potential. The fluorescent indicators were compared with replication competency, the conventional indicator of viability. By using these tools, the evaluation of the response of C. albicans to amphotericin B time-kill curves delineated four categories which may represent a continuum between alive and dead. The data showed that replication competency (CFU per milliliter) as determined by conventional antifungal susceptibility techniques provided only an estimate of inhibition. Interpretation of fluorescent staining characteristics indicated that C. albicans cells which were replication incompetent after exposure to greater than 0.5 microgram of amphotericin B per ml still maintained degrees of physiological function.

Comparison of three commercial systems for the identification of germ-tube negative yeast species isolated from clinical specimens.

Three commercial systems were evaluated for their ability to identify 171 germ-tube negative yeasts isolated from clinical specimens. The Yeast Biochemical Card and Analytical Profile Index 20 AUX identified 97% of 171 strains tested. The Biolog system had poor clinical utility: only 48% of strains were identified. For Yeast Biochemical Card and Analytical Profile Index 20 AUX, 9% and 6%, respectively, required repeat testing and both systems required supplemental tests for 28% of the strains. These observations indicate that considerable expertise and a battery of reagents in addition to the basic systems are required for accurate identification of germ-tube negative yeasts.

Evaluation of CHROMagar Candida for presumptive identification of clinically important Candida species.

CHROMagar Candida is a recently described and differential medium for the isolation and the presumptive identification of clinically important yeasts. We evaluated it with 262 yeast strains from clinical specimens, including 173 Candida albicans, 21 Candida tropicalis, 8 Candida krusei, 49 Candida glabrata, and 12 strains of other yeast species. Strains were presumptively identified on the basis of colony color and texture. These observations were compared with conventional identification results. Candida albicans was identified correctly in 170 (98%) of the 173 strains. A total of 46 of the 205 specimens that were plated on CHROMagar contained mixed cultures of yeast. Thirty-seven (80%) of these mixed cultures were not detected in the original specimens. CHROMagar Candida was useful for the rapid presumptive identification of Candida albicans and facilitated the recognition of mixed cultures. For other yeast species, it may provide additional information to laboratories that do not regularly perform identifications beyond the germ tube test.

Comparison of dose doubling with probenecid for sustaining serum cefuroxime levels.

Serum cefuroxime concentrations were measured over a 12 h period in ten healthy adults following three iv dosing regimens: 750 mg cefuroxime, 1.5 g cefuroxime, and 750 mg cefuroxime with 1 g of probenecid given orally 3 h before the cefuroxime infusion. Probenecid prolonged the serum cefuroxime half-life by 63% (P < 0.05) with a significant increase in the mean time for which serum cefuroxime concentrations exceeded the MIC90 for common respiratory pathogens (2 mg/L) compared with either 750 mg cefuroxime (2.2 h, P < 0.05) or 1.5 g of cefuroxime (0.9 h, P < 0.05) without probenecid. The cost of the 750 mg cefuroxime dose plus probenecid is approximately half that of a 1.5 g cefuroxime dose.

The ecology of anthrax spores: tough but not invincible.

Bacillus anthracis is the causative agent of anthrax, a serious and often fatal disease of wild and domestic animals. Central to the persistence of anthrax in an area is the ability of B. anthracis to form long-lasting, highly resistant spores. Understanding the ecology of anthrax spores is essential if one hopes to control epidemics. Studies on the ecology of anthrax have found a correlation between the disease and specific soil factors, such as alkaline pH, high moisture, and high organic content. Researchers initially suggested that these factors influenced vegetative anthrax bacilli. However, subsequent research has shown that vegetative cells of B. anthracis have very specific nutrient and physiological requirements and are unlikely to survive outside a host. Review of the properties of spores of B. anthracis and other Bacillus species suggests that the specific soil factors linked to epidemic areas reflect important environmental conditions that aid the anthrax spores in causing epidemics. Specifically, high levels of calcium in the soil may help to maintain spore vitality for prolonged periods, thereby increasing the chance of spores encountering and infecting a new host. Cycles of runoff and evaporation may collect spores dispersed from previous epidemics into storage areas, thereby concentrating them. Uptake of large doses of viable spores from storage areas by susceptible animals, via altered feeding or breeding behavior, may then allow the bacterium to establish infection and cause a new epidemic. Literature search for this review was done by scanning the Life Sciences Collection 1982-1994 using the keywords "anthrax" and "calcium and spore."

The use of bacteriophages to differentiate serologically cross-reactive isolates of Klebsiella pneumoniae.

In serological typing of Klebsiella pneumoniae strains from human, equine and environmental sources, the capsular identity of many isolates could not be determined because of serological cross-reactivity. A panel of 91 bacteriophages able to lyse each of the 77 capsular serotypes of K. pneumoniae was isolated and tested for the ability to distinguish between strains in a collection of 17 clinical isolates of K. pneumoniae which exhibited cross-reactivity with two or more capsular type sera. Most isolates could be assigned a capsular type by performing a simple streak test with bacteriophage, although some required the application of an efficiency of plating analysis to discern capsular type. Bacteriophage typing was found to be an effective, inexpensive and clinically practical adjunct to serotyping in distinguishing serologically cross-reactive K. pneumoniae isolates, irrespective of their origin.

Disk diffusion versus broth microdilution susceptibility testing of Haemophilus species and Moraxella catarrhalis using seven oral antimicrobial agents: application of updated susceptibility guidelines of the National Committee for Clinical Laboratory Standards.

Susceptibility testing of Haemophilus species and Moraxella catarrhalis is medium and inoculum dependent. Seven oral agents, ampicillin, amoxicillin-clavulanic acid, cefaclor, loracarbef, cefuroxime-axetil, cefixime, and erythromycin, were tested against 400 beta-lactamase-positive and -negative clinically significant respiratory strains of Haemophilus species and 100 strains of M. catarrhalis. Sources of the strains included teaching and regional hospitals and a private laboratory. All strains were tested by broth microdilution and disk diffusion in haemophilus test medium for Haemophilus species and Mueller-Hinton broth and agar for M. catarrhalis. Appropriate National Committee for Clinical Laboratory Standards (NCCLS) standards were followed. For Haemophilus species, by disk diffusion and broth microdilution, respectively, 27 and 27% of strains were resistant to ampicillin, 37 and 5% were resistant to erythromycin, 3 and 0.5% were resistant to cefaclor, 2 and 0.5% were resistant to loracarbef, and 0% were resistant to cefuroxime-axetil, cefixime, and amoxicillin-clavulanic acid. beta-Lactamase-negative ampicillin-resistant strains were not observed. Of M. catarrhalis strains, 56% were resistant to ampicillin by disk diffusion and 95% were resistant by broth microdilution. This species was susceptible to all other agents tested by either method. The disagreements between disk diffusion results and MICs for cefaclor, ampicillin, cefuroxime, and loracarbef that occurred with use of the 1990 NCCLS tables were resolved when the 1992 NCCLS tables were used.

Campylobacter fetus diarrhea in a Hutterite colony: epidemiological observations and typing of the causative organism.

Following a case of Campylobacter fetus sepsis and meningitis in a 4-month-old female member of a Hutterite colony, an epidemiological investigation revealed at least 18 cases of diarrhea in other members of the colony. C. fetus was isolated from 7 of 15 fecal samples submitted from affected persons. A case control study suggested that persons who worked in the abattoir were 2.03 times more likely to have had diarrhea, but none of the risk factors studied were significant. The epicurve of the outbreak was inconclusive as to the likely mode of spread of C. fetus. All of the C. fetus strains isolated from the blood of the infant and from the fecal samples were the same by biochemical and antibiotic susceptibility tests. Pulsed-field gel electrophoresis showed that all isolates produced identical restriction endonuclease patterns and differed from other nonepidemiologically related strains of C. fetus.

Klebsiella pneumoniae infection on a rehabilitation unit: comparison of epidemiologic typing methods.

OBJECTIVE: To identify factors associated with an increased occurrence of Klebsiella pneumoniae isolation in urine cultures and infected wounds on a rehabilitation unit and to compare typing methods for K pneumoniae isolates. DESIGN: Retrospective review of laboratory reports and patient records with case-control study. Analysis of K pneumoniae isolates using capsular serotyping, enzyme electrophoretic typing, ribotyping, and DNA typing. SETTING: 48-bed rehabilitation unit in an 1,100-bed tertiary care teaching hospital in Winnipeg, Manitoba. RESULTS: In 1988, 20 (19%) of 106 patients admitted to the rehabilitation unit had K pneumoniae isolated from urine or wound, and in 1989 31 (28%) of 111 patients had Klebsiella isolated. Review of ward practices revealed appropriate written policies but evidence of failure in execution leading to multiple opportunities for transmission among patients. Substantial environmental contamination was not identified, although a common urine graduate may have contributed to some transmission. Individuals with K pneumoniae isolated had a significantly longer duration of stay. Many of these were spinal cord-injured patients and were maintained on intermittent catheterization. One outbreak strain was identified in epidemiologic typing. Other strains were generally identified in individuals with non-nosocomial acquisition of infection. Comparison of epidemiologic typing methods suggests ribotyping may be the optimal method for typing K pneumoniae strains. CONCLUSIONS: K pneumoniae was acquired frequently by spinal cord-injured patients with extended admissions, re-emphasizing the importance of both patients and staff following appropriate infection control practices on rehabilitation wards. Ribotyping was the optimal method for typing K pneumoniae isolates.

Klebsiella pneumoniae gastroenteritis masked by Clostridium perfringens.

An unusual food-borne outbreak of gastroenteritis associated with contaminated turkey occurred at a catered company meal. The average incubation period was 10 h, and the predominant symptoms were watery diarrhea and cramps. Vomiting did not occur. Initial epidemiological features and cultures from turkey and feces of infected patients suggested that the causative agent was Clostridium perfringens, but Klebsiella pneumoniae of capsular type K15 was also isolated in large numbers from both the turkey and feces of the same patients. Plasmid analysis and enterotoxin results supported the role of K. pneumoniae as the causative agent in this outbreak. Organisms other than commonly identified pathogens should not be ignored if present in high concentrations in both food and feces of infected persons.