Letitia C. Fulcher

Susceptibility of Acinetobacter Strains Isolated from Deployed U.S. Military Personnel

ABSTRACT The susceptibilities of 142 Acinetobacter baumannii-calcoaceticus complex isolates (95 from wounded U.S. soldiers deployed overseas) to 13 antimicrobial agents were determined by broth microdilution. The most active antimicrobial agents (≥95% of isolates susceptible) were colistin, polymyxin B, and minocycline.

17α -Substituted analogs of estradiol for the development of fluorescent estrogen receptor ligands

For the successful development of a high-affinity fluorophore-estradiol conjugate, the fluorophore must be attached to the estradiol molecule at a position that interferes least with its binding to the receptor. We have concentrated on 17 alpha substituents as models for fluorophore attachment, based on literature precedent and on our earlier work with small 17 alpha side chains. In this report, we describe syntheses and estrogen receptor binding affinities of 19 analogs of estradiol substituted in the 17 alpha position with larger side chains (of six to 11 carbons), some of which may be synthetically modified to link a fluorophore. These analogs were synthesized either by nucleophilic cleavage of estrone-17 beta-oxirane 3-benzyl ether and subsequent debenzylation (4 to 18), by cross-coupling of alkynes (21 to 24), by alkylation of 17 alpha-ethynylestradiol 3,17-bis(tetrahydropyranyl ether) and subsequent acidic hydrolysis (25 to 28), or by reacting estrone either with appropriate aryl/alkynyllithium reagents (29, 30, and 32) or with benzylmagnesium bromide (31). Relative binding affinities of these newly synthesized analogs were determined for estrogen receptor (rat uterus) using a standard competition assay. The results suggest that analogs with reduced mobility and/or more polarizable electron density in the side chain generally bind more strongly to the receptor. The relative affinities of several selected compounds were also determined in the presence of 4% dimethylformamide; some compounds bearing larger, nonpolar 17 alpha substituents showed dramatically improved affinities, while affinities for compounds with shorter nonpolar side chains remained largely unchanged. These binding affinity results should be useful in designing new high-affinity fluorescent ligands for the estrogen receptor.

Detection of CTX-M-Type Extended-Spectrum Beta-Lactamase (ESBLs) by Testing with MicroScan Overnight and ESBL Confirmation Panels

ABSTRACT CTX-M extended-spectrum beta-lactamases (ESBLs) have emerged as the most common type of ESBL globally, their incidence easily surpassing those of SHV and TEM ESBLs in most locales. This study compared the performance of two MicroScan dried panels with CLSI reference broth microdilution and disk diffusion methods on a collection of genetically characterized ESBL-producing isolates. These included 64 Enterobacteriaceae isolates that produced CTX-M8, -14, -15, or -16 according to PCR and sequencing of the bla gene, 17 isolates that produced a SHV or TEM ESBL, and 19 that produced both CTX-M and SHV ESBLs. Each isolate was tested by a frozen reference microdilution panel, the MicroScan ESβL plus confirmation panel, and a routine dried panel containing streamlined ESBL confirmation dilutions (MicroScan Neg MIC panel type 32) that included cefotaxime and ceftazidime tested alone or with a fixed concentration of 4 μg/ml of clavulanate. Each isolate was also tested by the standard CLSI double-disk confirmation tests. The disk diffusion method detected all ESBL-producing isolates, the frozen reference panel detected 90% of isolates (10 out of 100 could not be analyzed because of off-scale MICs that exceeded the clavulanate combination concentrations in the panel), the ESβL plus panel detected 98% (1 missed and 1 off scale), and the streamlined ESBL panel detected 95% (5 off scale). Very high MICs for a few strains that produced SHV or both CTX-M and SHV ESBLs precluded noting the required three twofold-dilution differences with clavulanate needed to confirm an ESBL primarily in the reference panel and the Neg type 32 panel.

Multilaboratory Evaluation of Disk Diffusion Antimicrobial Susceptibility Testing of Neisseria meningitidis Isolates

ABSTRACT In 2005, the Clinical and Laboratory Standards Institute published MIC interpretive criteria for 13 antimicrobial agents used for either therapy or prophylaxis of Neisseria meningitidis infections. The MIC method includes the use of lysed horse blood-supplemented Mueller-Hinton broth with incubation in 5% CO2 for 20 to 24 h. Since some clinical laboratories might prefer the option of disk diffusion testing for infrequently encountered isolates a multicenter collaborative study was conducted to evaluate the reproducibility of a disk diffusion method for testing isolates of N. meningitidis. Interpretive criteria were developed for 12 antimicrobial agents. Four laboratories tested a common collection of 50 meningococcal strains and then tested 25 unique isolates per laboratory. Isolates were tested using Mueller-Hinton sheep blood agar plates incubated for 20 to 24 h in 5% CO2; they were also tested by the reference broth microdilution method in parallel. Pooling of the MIC and disk diffusion data from the common and unique isolates provided a sufficient sample size to develop susceptible, intermediate, and resistant zone diameter interpretive criteria using the error rate-bounded method for the following agents: chloramphenicol, trimethoprim-sulfamethoxazole, ciprofloxacin, and rifampin. Due to the lack of resistant strains at the present time, “susceptible only” interpretive criteria were proposed for cefotaxime, ceftriaxone, meropenem, azithromycin, and minocycline. The numbers of minor interpretive errors with penicillin and ampicillin disk tests were unacceptably high and precluded recommended testing of those agents by the disk method. However, amdinocillin, an agent that preferentially binds to the altered penicillin binding protein responsible for diminished penicillin susceptibility, has potential utility as a surrogate screening reagent for ampicillin resistance. A disk diffusion breakpoint was derived for nalidixic acid to serve as a surrogate marker for gyrase A mutations associated with diminished fluoroquinolone susceptibility. Disk diffusion testing with meningococci can be performed in a reproducible manner with several antimicrobial agents and represents a practical and cost-effective option for testing sporadic clinical isolates or for surveillance purposes by resource-limited laboratories.

Evaluation of Disk Approximation and Single-Well Broth Tests for Detection of Inducible Clindamycin Resistance in Streptococcus pneumoniae

ABSTRACT This study evaluated an agar disk diffusion D-zone test and an erythromycin-clindamycin (ERY + CLI) single-well broth test for inducible CLI resistance in Streptococcus pneumoniae. The standard CLSI disk approximation test and a single-well combination test incorporating 1 plus 0.5 μg/ml ERY + CLI detected >96% of isolates containing the ermB determinant.

Development of Doxycycline MIC and Disk Diffusion Interpretive Breakpoints and Revision of Tetracycline Breakpoints for Streptococcus pneumoniae

ABSTRACT A study was performed to derive susceptibility testing interpretive breakpoints for doxycycline with Streptococcus pneumoniae and to reassess breakpoints for tetracycline using the requirements defined in Clinical and Laboratory Standards Institute (CLSI) document M23-A3. Tetracycline and doxycycline MICs and disk diffusion zone sizes were determined on 189 isolates selected from the 2009-2010 CDC Active Bacterial Core surveillance strain collection according to the testing methods described in CLSI documents M07-A8 and M02-A10. Tetracycline and doxycycline MICs and zones were compared to each other directly, and the reproducibility of MICs and zone diameters for both drugs was determined. Scattergrams of tetracycline MICs versus corresponding zone diameters and doxycycline MICs versus zones were prepared, and analysis indicated that the present CLSI tetracycline MIC and disk breakpoints did not fit the susceptibility data for doxycycline. Doxycycline was 1 to 3 dilutions more potent than tetracycline, especially in strains harboring the tetM resistance determinant. tetM was detected in ≥90% of isolates having tetracycline MICs of ≥4 μg/ml and in ≥90% with doxycycline MICs of ≥1. Limited pharmacokinetic/pharmacodynamic (PK/PD) data coupled with application of the error-rate bounded method of analysis suggested doxycycline-susceptible breakpoints of either ≤0.25 μg/ml or ≤0.5 μg/ml, with intermediate and resistant breakpoints 1 and 2 dilutions higher, respectively. The disk diffusion zone diameter correlates were susceptible at ≥28 mm, intermediate at 25 to 27 mm, and resistant at ≤24 mm. Revised lower tetracycline MIC breakpoints were suggested as susceptible at ≤1 μg/ml, intermediate at 2 μg/ml, and resistant at ≥4 μg/ml. Suggested tetracycline disk diffusion zones were identical to those of doxycycline.

Detection of Inducible Clindamycin Resistance in Beta-Hemolytic Streptococci by Using the CLSI Broth Microdilution Test and Erythromycin-Clindamycin Combinations

ABSTRACT This study assessed an erythromycin-clindamycin (ERY-CC) broth test for inducible CC resistance in beta-hemolytic streptococci. One hundred one isolates of groups A, B, C, F, and G were tested by the CLSI broth microdilution method. Combinations of 1 and 0.25 μg/ml or 0.5 and 0.25 μg/ml of ERY and CC, respectively, detected all inducible isolates.

Detection of Favorable Oral Cephalosporin-Clavulanate Interactions by In Vitro Disk Approximation Susceptibility Testing of Extended-Spectrum-Beta-Lactamase-Producing Members of the Enterobacteriaceae

ABSTRACT Extended-spectrum-beta-lactamase (ESBL)-producing members of the Enterobacteriaceae are often resistant to multiple drug classes, making therapy of urinary infections with oral antibiotics difficult. Previously it was shown that amoxicillin-clavulanate can provide clavulanate inhibition of ESBLs and protect an oral cephalosporin present in combination when tested by broth microdilution. This study has shown that disk approximation testing could detect favorable cephalosporin-clavulanate interactions among a group of 101 previously characterized members of the Enterobacteriaceae with CTX-M, SHV, or TEM ESBLs.

Collaborative Evaluation of an Erythromycin-Clindamycin Combination Well for Detection of Inducible Clindamycin Resistance in Beta-Hemolytic Streptococci by Use of the CLSI Broth Microdilution Method

ABSTRACT Constitutive or inducible clindamycin resistance can occur in beta-hemolytic streptococci due to the presence of an erm gene. The Clinical and Laboratory Standards Institute (CLSI) has recommended a disk approximation test (D-zone test) with erythromycin and clindamycin disks and a single-well broth test combining erythromycin and clindamycin for detection of inducible clindamycin resistance in staphylococci, but only a disk approximation test for the beta-hemolytic streptococci. This collaborative study assessed two different erythromycin and clindamycin concentration combinations in single wells (1 μg/ml + 0.25 μg/ml [erythromycin plus clindamycin] and 1 μg/ml + 0.5 μg/ml) with three different brands of Mueller-Hinton broth supplemented with 3% lysed horse blood for testing of frozen panels prepared for this study. All labs performed the D-zone test as described by the CLSI. A total of 155 nonduplicate streptococcal isolates (50 group A, 48 group B, 28 group C, and 29 group G isolates) were tested; 99 isolates showed inducible resistance by the D-zone test. There were some differences noted based upon the test medium. The sensitivity of the erythromycin plus clindamycin combination of 1 μg/ml + 0.25 μg/ml was 91 to 100%, while the sensitivity of the combination of 1 μg/ml + 0.5 μg/ml was 95 to 100%. Specificity overall was 98%. The slightly higher sensitivity of the combination of 1 μg/ml + 0.5 μg/ml is recommended. This study has demonstrated that a single-well microdilution test incorporating erythromycin and clindamycin in combination is a sensitive and specific indicator of inducible clindamycin resistance and could be included in routine test panels.

Potency and Sterility of Fortified Tobramycin, Fortified Vancomycin, and Moxifloxacin at 4, 24, and 35°C for 14 Days.

Purpose: To assess the potency and sterility of ophthalmic antibiotic drops commonly used in the treatment of bacterial keratitis. Methods: This was a basic investigation. Three drugs were tested: fortified vancomycin 25 mg/mL, fortified tobramycin 14 mg/mL, and moxifloxacin 5 mg/mL. A bottle of each was stored separately at 4, 24, and 35°C, with the potency determined by microbiological assay at 0, 7, and 14 days. Differences in potency were assessed by 2-way analysis of variance followed by a 1-way repeated-measures analysis of variance with Bonferroni post hoc testing as warranted. Sterility of drugs when handled by patients for varying periods was confirmed by culturing samples on MacConkey and sheep blood agars. Results: The concentration of fortified tobramycin and moxifloxacin remained constant over 14 days at the 3 tested temperatures. The concentration of fortified vancomycin remained constant at 4°C, but it declined by 38% ± 1% (P = 0.001) at 24°C on day 14 and by 48% ± 1% (P = 0.001) and 78% ± 3% (P = 0.0009) at 35°C on days 7 and 14, respectively. A total of 49 drops (mean, 7.3 days; range, 1–18 days) were tested for sterility, and all were negative for microbial contamination. Conclusions: All 3 drugs remained potent at 4°C for up to 14 days. Fortified tobramycin and moxifloxacin also maintained potency for 14 days at 24 and 35°C. In contrast, fortified vancomycin lost its potency by day 14 at 24°C and by day 7 at 35°C. All in-use antibiotic drops tested were sterile. The results indicate that patients should be cautioned to store vancomycin under refrigerator or at least under cool conditions.