Biomedical subjects
B F Woolfrey
Publications and source records attributed to B F Woolfrey.
Evaluation of oxacillin tolerance in Staphylococcus aureus by a novel method.
A novel agar dilution plate-count procedure for the quantitative measurement of bacterial inhibition and killing is described. For Staphylococcus aureus versus oxacillin, by the agar dilution plate-count procedure it was found that only 1 of 20 clinical isolates and 1 of 7 allegedly tolerant reference isolates met the conventional definition of tolerance. By using inocula of 10(5) CFU per plate, most isolates were demonstrated to have subpopulations of cells which, although inhibited, persisted for 24 h in concentrations significantly above their MICs. The persister percentages at 24 h appeared to be strain dependent, and all persisters exhibited the paradoxical effect. For each isolate, the persister percentage markedly decreased after action by oxacillin for 48 h, and the paradoxical effect was greatly diminished. Our findings suggest that tolerance is an artificial and arbitrary concept that does not adequately characterize the inhibition and killing dynamics associated with the persister phenomenon.
Evaluation of mannitol salt agar with oxacillin as a screening medium for methicillin-resistant Staphylococcus aureus.
We evaluated the use of mannitol salt agar with oxacillin for use as a primary screening medium for the simultaneous detection and identification of methicillin-resistant Staphylococcus aureus in clinical surveillance specimens. Oxacillin agar dilution susceptibility tests with mannitol salt agar and Mueller-Hinton agar were performed in parallel with disk-agar diffusion testing on 95 oxacillin-susceptible and 105 oxacillin-resistant S. aureus stock isolates. MICs were found to be comparable, showing distinct separation of susceptible and resistant isolates into two groups with MICs of less than or equal to 2 and greater than or equal to 32 micrograms/ml, respectively. In accord with these findings, 4 micrograms of oxacillin per ml was selected for use in the screening medium. For performance evaluation, mannitol salt agar with 4 micrograms of oxacillin per ml was compared with mannitol salt agar without oxacillin by performing parallel screening tests on 153 clinical surveillance specimens. For detection of methicillin-resistant S. aureus, mannitol salt agar with 4 micrograms of oxacillin per ml was as sensitive as mannitol salt agar without oxacillin and required significantly fewer confirmatory tests. For primary identification of methicillin-resistant S. aureus, mannitol salt agar with 4 micrograms of oxacillin per ml was 6.4% false-positive and 1.1% false-negative, with a 93.6% positive predictive value. These findings indicate that mannitol salt agar with 4 micrograms of oxacillin per ml can be used as a reliable and cost-effective screening medium for the simultaneous detection and identification of methicillin-resistant S. aureus in clinical surveillance specimens.
Pasteurella multocida in an infected tiger bite.
We report an unusual case of Pasteurella multocida wound infection caused by a tiger bite. We investigated the normal fang flora of large zoo cats and found P multocida in cultures from seven tigers, three of four leopards, and one lynx. Sucrose fermentation was found to be highly media dependent and unpredictable. The literature relative to P multocida in bite-wound infections is reviewed with special reference to bites by animals other than cats and dogs. With the addition of the present case, the animals involved have been two rats, two opossums, two lions, one horse, one rabbit, one boar, one panther, and one tiger.
Overutilization of cultures of CSF for mycobacteria.
Over a five-year period, 1,883 specimens were cultured for mycobacterium tuberculosis (TB) at our hospital. All cultures were negative, and no cases of tuberculous meningitis were diagnosed. Culture rates (percent of CSF specimens cultured for TB) varied from 74% on the Medicine and Neurology services to 6% on the Pediatric service. These culture rates have been stable for five years. These data suggest that on the Medicine and Neurology services, TB cultures of CSF are persistently overutilized. A simple rule of not culturing CSF for TB if results of the CSF analysis are normal (ie, WBC count less than or equal to 4/cu mm, protein level less than or equal to 45 mg/dL, and glucose level greater than or equal to 45 mg/dL) could reduce utilization by at least 50% without adversely affecting quality of care.
An evaluation of three rapid coagglutination tests: Sero-STAT, Accu-Staph, and Staphyloslide, for differentiating Staphylococcus aureus from other species of staphylococci.
Three commercial coagglutination tests--Sero-STAT, Accu-Staph, and Staphyloslide--were performed in parallel with slide coagulase, tube coagulase, and thermostable nuclease tests on 100 methicillin-susceptible Staphylococcus aureus (MSS) strains, 100 methicillin-resistant S. aureus (MRS) strains, and 100 non-S. aureus staphylococcal strains (NSA). All three coagglutination tests showed sensitivities of 100% for MSS strains. For MRS strains, sensitivities were, respectively, 99%, 100%, and 99%. False-positive reactions were, respectively, 10%, 2%, and 2%. A marked difference in slide coagulase test sensitivity was found for MSS strains (79%) and MRS strains (14%). These findings suggest that the coagglutination tests may be less sensitive for detecting MRS strains than for detecting MSS strains and that these properties may be related to clumping factor reactivity. The high false-positive rate for Sero-STAT and even the 2% false-positive rate for Accu-Staph and Staphyloslide make clinical usefulness at this time somewhat problematic and debatable. In view of these findings, the authors prefer to retain the tube coagulase test and thermostable nuclease test for differentiation of S. aureus from non-S. aureus strains in their laboratory.
Antimicrobial resistance in Haemophilus isolates: a Minnesota experience and literature review.
Annual ampicillin susceptibility rates for Haemophilus influenzae isolates at the St. Paul-Ramsey Medical Center gradually decreased from 100% in 1974 to 83.3% in 1980 and then remained stable at 88.90%. Penicillin susceptibility rates were similar to those for ampicillin. Ampicillin rates were source dependent: eye 95%, respiratory 90%, miscellaneous sources 82%, and blood and CSF 80%. Rates for Haemophilus parainfluenzae varied and showed no trend. H. parainfluenzae isolates were distinctly less susceptible to penicillin (70%) than to ampicillin (96%). H. influenzae isolates were highly susceptible to chloramphenicol (99.6%) and tetracycline (97.5%), with the latter also showing source dependency. Characterization of isolates for colony morphology and hemolysis showed no clinical relevancy. Ampicillin and penicillin MICs were determined for 128 clinical isolates saved in stock culture during 1978-1983. All 19 resistant isolates (MIC greater than or equal to 4 micrograms/mL) were resistant to both penicillin and ampicillin and produced beta-lactamase. Eight had penicillin MICs of 1 or 2 micrograms/mL and three had ampicillin MICs of 1 or 2 micrograms/mL. The significance of isolates with MICs of 1-2 micrograms/mL is discussed in relation to our findings and a review of the literature.
Evaluation of the automicrobic system for detection of resistance of Staphylococcus aureus to methicillin.
The AutoMicrobic system (AMS) (Vitek System, Inc., Hazelwood, Mo.) was tested for its ability to determine oxacillin and gentamicin susceptibility of 98 known oxacillin-susceptible and 103 known oxacillin-resistant Staphylococcus aureus isolates. AMS and reference oxacillin susceptibility results were in agreement for all 95 (100%) oxacillin-susceptible isolates. In contrast, only 23 (22.3%) of the 103 known oxacillin-resistant isolates were correctly reported. For the known oxacillin-resistant isolates, 65 received AMS reports at 3 to 4 h, with only 9% being correct, whereas 38 were reported at 5 to 6 h, with 47% being correct. The reliability of AMS gentamicin susceptibility results was evaluated by testing the 198 S. aureus isolates in parallel with MIC-2000 broth dilution tests. AMS gentamicin susceptibility results were found to be reliable and essentially identical to MIC-2000 results. The possibility of improving AMS oxacillin resistance detection by using gentamicin resistance as a linked screening marker for oxacillin resistance was evaluated with data from the parallel AMS and MIC-2000 gentamicin susceptibility tests and from data accrued on recent clinical laboratory isolates. By these two approaches, respective sensitivities of 97 and 99.8%, and specificity of 72%, were found for detection of oxacillin-resistant isolates by using gentamicin resistance as a marker.
Evaluation of the automicrobic system for susceptibility testing of Pseudomonas aeruginosa to gentamicin, tobramycin, and amikacin.
The AutoMicrobic system (AMS; Vitek Systems, Inc., Hazelwood, Mo.) was studied for its ability to produce accurate and precise MIC interpretations for Pseudomonas aeruginosa susceptibility to gentamicin, tobramycin, and amikacin. MICs were determined in parallel on 200 selected P. aeruginosa isolates by using the AMS discrete-integer MIC program AMS p12.ROB for interpretation of the AMS Gram-Negative General Susceptibility Urinary Card, and a reference small-integer broth microdilution test. Parallel AMS and broth microdilution MICs were also replicated for three selected strains of P. aeruginosa for which MICs were representative of the dilution test ranges. For the 200 P. aeruginosa isolates, mean AMS MICs were significantly larger than the reference test mean MICs, coefficients of variation were approximately double those of the reference test, and correlation coefficients were unacceptably low for each antimicrobial agent. MIC replication studies for the three selected P. aeruginosa strains showed comparable AMS and reference mean MICs in the lower portions of the dilution ranges, significantly higher AMS mean MICs in the upper portions, and mean coefficients of variation of 63 and 9.6%, respectively, for replicated AMS and reference MICs. These results indicate that the AMS, in its present stage of development, does not produce acceptable MIC measurements for P. aeruginosa susceptibility to gentamicin, tobramycin, and amikacin.
Evaluation of the AutoMicrobic system for identification and susceptibility testing of gram-negative bacilli.
The AutoMicrobic system (AMS) (Vitek Systems, Inc., Hazelwood, Mo.) was compared with the API-20E system for the identification of gram-negative bacilli by using 380 stock clinical isolates and 377 immediately encountered fresh clinical isolates. For the stock isolates, with Enterobacteriaceae-Plus Biochemical Cards and automated interpretation, 364 (95.8%) were in agreement to the species level. For the fresh clinical isolates, agreement at the genus and species levels was 89.7 and 85.9%, respectively, when Enterobacteriaceae-Plus Cards were interpreted by the AMS. Manual interpretation of Enterobacteriaceae-Plus Biochemical Cards improved species level agreement to 91.0%. Subsequent retesting of all discrepant isolates with the Gram-Negative Identification Card resulted in significant improvement of results, and for the stock and fresh clinical isolates, species level agreement was 98.7 and 97.3%, respectively. AMS susceptibility testing was evaluated by comparing ampicillin and cephalothin MICs determined in parallel by AMS and a reference broth microdilution test for stock isolates, and by comparison of AMS and standardized disk agar diffusion test results for fresh clinical isolates. For the stock isolates, AMS mean integer MICs approximated microdilution mean integer MICs with AMS, providing excellent MIC replicability. For ampicillin and cephalothin, 50 and 46.8%, respectively, of AMS integer MICs were within +/- 1 microgram/ml of the reference values, and 89.3 and 63.1% of AMS integer MICs were within +/- 2 micrograms/ml of the reference values. For the fresh clinical isolates, AMS and reference results were in disagreement for 4.5% of the antimicrobial agents tested, with 2.3% as a combination of "major" and "very major" errors.
Error rates associated with the use of recently proposed breakpoints for testing Pseudomonas aeruginosa versus gentamicin, tobramycin, and amikacin by the standardized disk agar diffusion test.
Two hundred fifteen Pseudomonas aeruginosa isolates were tested in parallel by the disk agar diffusion test, using a standardized agar preparation, and by a microbroth test, using dilutions differing by small arithmetic increments. For gentamicin, recently proposed breakpoints of resistance (R) less than or equal to 12 mm and susceptibility (S) greater than or equal to 16 mm produced error rates of 20 and 6.8%, respectively. Limiting the error rate for susceptible interpretations to less than or equal to 2% produced a widening of the intermediate zone to include 67.4% of the isolates tested. For tobramycin, the recently proposed breakpoints of R less than or equal to 12 mm and S greater than or equal to 15 mm were associated with error rates of 66.7 and 1.4%, respectively. Breakpoints of R less than or equal to 12 mm and S greater than or equal to 13 mm were demonstrated to be equally effective when the error rate for susceptible interpretations was limited to less than or equal to 2% by error rate-bound analysis. For amikacin, proposed breakpoints of R less than or equal to 14 mm and S greater than or equal to 17 mm were associated with error rates of 27.3 and 3.2%, respectively. Limiting the error rates for susceptible interpretations to less than or equal to 2% required breakpoints of R less than or equal to 14 mm and S greater than or equal to 18 mm. The ability to establish effective susceptibility breakpoints for tobramycin and amikacin appeared not to be related to the disk agar diffusion test process itself but rather to the high degree of susceptibility of the P. aeruginosa population. These findings severely limit the usefulness of the disk agar diffusion procedure for testing P. aeruginosa versus the aminoglycosides. For this purpose, we recommend dilution tests which employ small arithmetic increment schemes.
Comparative evaluation of the micro-media system, sceptor, and MIC-2000 microdilution methods for testing Pseudomonas aeruginosa against gentamicin, tobramycin, and amikacin.
Minimum inhibitory concentrations (MICs) for selected strains of Pseudomonas aeruginosa versus gentamicin, tobramycin, and amikacin were replicated in parallel with MMS (Micro-Media Systems, Potomac, Md). Sceptor (BBL Microbiology Systems, Cockeysville, Md), and matching MIC-2000 (Dynatech Laboratories, Inc., Alexandria, Va.) twofold dilution panels and with MIC-2000 panels with dilutions differing by small arithmetic increments. The three microdilution systems produced comparable modal MICs. However, dispersion about modal values was greater for MMS than for either Sceptor or comparable MIC-2000 twofold panels. MICs were best define by MIC-2000 panels with dilutions differing by small arithmetic increments, for which 72.9% of MICs were modal, 95.4% were one small increment step or less from the modal value, and 100% were two small-increment steps or less form the modal value. The similarity of MIC replication for Sceptor and MIC-2000 twofold panels suggests the possibility of using small increment dilutions by Sceptor.
Evaluation of the AutoSCAN-3 and Sceptor systems for Enterobacteriaceae identification.
To evaluate the accuracy and cost effectiveness of the AutoSCAN-3 (Micro-Scan Systems of America, Sacramento, Calif.) and Sceptor (BBL Microbiology Systems, Cockeysville, Md.) systems for identification of members of the Enterobacteriaceae, we performed parallel tests on 678 stock cultures of well-characterized clinical isolates of Enterobacteriaceae. Automated results by AutoSCAN-3 correctly identified 95.1% at the genus level and 94.9% at the species level. However, 15 of 42 Shigella isolates were misidentified as members of other genera. In contrast to the automated results, visual interpretation of panels produced 97.9% agreement at the genus level, missing only three Shigella isolates. Sceptor correctly identified 96.8% at the genus level and 93.4% at the species level. Of 42 Shigella isolates, 3 were missed and were designated as Salmonella spp. Although all Salmonella spp. were correctly identified, six other isolates were misidentified as Salmonella spp. Test costs were found to be comparable for each system, with the cost per test increasing markedly with fewer than 10 to 15 tests performed per day.
Broth microdilution testing of Pseudomonas aeruginosa and aminoglycosides: need for employing dilutions differing by small arithmetic increments.
The use of dilutions differing by small arithmetic increments was studied as a means for improving the definition and measurement of minimum inhibitory concentrations and precision parameters for testing Pseudomonas aeruginosa versus the aminoglycosides by the broth microdilution test. For five strains of P. aeruginosa versus gentamicin, tobramycin, and amikacin, comparisons were made of minimum inhibitory concentrations which were replicated in parallel by using three microdilution systems: small increment panels prepared by us, modified twofold dilution panels prepared by us, and similar modified twofold dilution panels obtained commercially. The small increment dilutions were prepared to differ by concentrations of 1.0 microgram/ml for gentamicin and tobramycin and by 2.0 micrograms/ml for amikacin. Use of the small increment dilutions resulted in the ability to measure minimum inhibitory concentrations at more closely spaced intervals than those dictated by modified twofold dilution schemes, and confidence limits were significantly improved. The average coefficient of variation for the small increment microdilution test results was 9.5%, with 99.5% of minimum inhibitory concentrations falling within +/- 2 small increment dilutions from their modal values.
Comparison of minimum inhibitory concentration values determined by three antimicrobic dilution methods for Pseudomonas aeruginosa.
This investigation compares minimum inhibitory concentration measurements by three antimicrobic dilution methods for Pseudomonas aeruginosa versus seven antimicrobics. Minimum inhibitory concentrations were measured for 650 P. aeruginosa clinical isolates and for repeated tests with P. aeruginosa ATCC 27853 versus gentamicin, tobramycin, amikacin, netilmicin, sisomicin, carbenicillin, and ticarcillin, using the macro-broth, micro-broth, and agar dilution methods. For all antimicrobics, it was found that the micro-broth and agar dilution methods produced comparable minimum inhibitory concentration measurements, which were found to lie 1 to 2 double dilution steps below those determined by the macro-broth method. Acceptably replicability was found for both the macro-broth and the agar dilution methods. The micro-broth method showed less replicability, with 4.7% of minimum inhibitory concentration values lying +/- 2 or more double dilution steps from the modal value. It is important to recognize such differences if micro-broth or agar dilution methods are substituted for the macro-broth method.
Inability of the API-20E system to speciate the fluorescent group of pseudomonads.
One thousand randomly selected clinical isolates of the fluorescent group of Pseudomonas were speciated in parallel by the API-20E system and by a classic microbiologic 17-test battery. The classic battery identified the isolates as 993 Pseudomonas aeruginosa, five Pseudomonas putida, and two Pseudomonas fluorescens. To augment the P. putida and P. fluorescens data, 52 reference isolates were also tested in parallel. API-20E was found to identify 99% of P. aeruginosa as P. aeruginosa. However, the 59 P. putida and P. fluorescens isolates were not accurately identified and were designated 58% P. aeruginosa, 24% P. stutzeri, 16% ambiguous categories, and only 2% in agreement with the classic designation. These findings indicated that the API-20E system does not accurately identify the non-P. aeruginosa fluorescent pseudomonads and that errors in P. aeruginosa designations will increase as the percentages of P. putida and P. fluorescens increase in the population sample.
An evaluation of burn wound quantitative microbiology. I. Quantitative eschar cultures.
The reliability of quantitative data from burn wound biopsy cultures was investigated. This was done by comparing the recovery of microorganisms from a series of burn wound eschar biopsy specimens that were each divided into two approximately equal portions and cultured in parallel. The results indicate that a microorganism present in the burn wound site in any quantity has at least a 25% chance of being missed by a single quantitative eschar culture. For recovery levels corresponding to quantitative breakpoints that have been proposed to be predictive of burn wound sepsis, only 38% of paired quantitative results agreed within the same log10 unit, and 44% differed by +/- 2 log10 units or more. These findings indicate that quantitative results derived from burn wound biopsy cultures are unreliable and may be significantly misleading when used for decision-making relative to patient care.
An analysis of error rates for disc agar-diffusion testing of Pseudomonas aeruginosa versus aminoglycosides.
Five hundred thirty-five recent clinical isolates of Pseudomonas aeruginosa were tested in parallel by the standardized disc agar-diffusion test and a micro-broth dilution test to evaluate the error rates associated with the use of both fixed indeterminate-zone breakpoints and floating indeterminate-zone breakpoints for assessing susceptibility to amikacin, gentamicin, and tobramycin. Error rate-bound analysis showed that unacceptably high rates of error were associated with the use of all fixed breakpoints. Error rates were improved when the floating indeterminate-zone principle was used, but rates still remained unacceptably high. These findings indicate the disc agar-diffusion test should not be used for testing the susceptibility of P. aeruginosa to the aminoglycosides.