Meredith E. Erwin

Antimicrobial activity and spectrum of SCH27899 (ziracin®) tested against gram-positive species including recommendations for routine susceptibility testing methods and quality control

Abstract SCH27899 is an oligosaccharide, everninomicin antibiotic with activity primarily against Gram-positive pathogens. The activity of SCH27899 was evaluated against 360 routine clinical isolates by the broth microdilution (BMD), agar dilution (AD), disk diffusion (DD), and Etest (AB BIODISK, Solna, Sweden) methods. In addition, results from a nine center SCH27899 quality control (QC) trial were used to establish QC ranges. SCH27899 MICs for 330 Gram-Positive strains, including multiply-resistant staphylococci and enterococci, ranged from 0.015 to 1 μg/ml with MIC 90 s of 0.12 to 0.5 μg/ml. SCH27899 had no measurable activity against the 30 selected Gram-negative strains tested (MICs, > 256 μg/ml), with the exception of Moraxella catarrhalis MICs, 0.12 μg/ml). Etest MICs for SCH27899 correlated well with AD and BMD results with > 90% of MICs within ± one log 2 dilutions of the reference test results. Three disk concentrations (2.5-, 5-, 10-μg) of SCH27899 were evaluated, but minimal difference of zone diameters between disk drug contents was observed (± 2 mm). SCH27899 disk zone diameters correlated poorly with reference MICs due to small zone diameters (range, 11 to 22 mm) attributed to poor diffusion through agar mediums, a product of this compounds high molecular weight and solubility. The use of the DD method for SCH27899 was not recommended. The proposed MIC quality assurance limits for SCH27899 using Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 was 0.06 to 0.25 μg/ml for both QC strains and methods. SCH27899 appears to be a eveminomicin-derivative widely active against important Gram-positive cocci, and in vitro dilution testing methods would be preferred for clinical use, validated by the recommended MIC control ranges cited in this report.

Comparative antimicrobial activity of gatifloxacin tested against Streptococcus spp. including quality control guidelines and etest method validation

Gatifloxacin (formerly AM-1155 or CG 5501) is a new 8-methoxy fluoroquinolone with enhanced activity against Gram-positive cocci, especially Streptococcus pneumoniae and other streptococci. Recent clinical strains (599 isolates) were tested against gatifloxacin, three comparison fluoroquinolones, and penicillin by the reference broth microdilution, Etest (AB BIODISK, Solna, Sweden) and standardized disk diffusion methods (5 micrograms gatifloxacin disk). Gatifloxacin (MIC90, 0.5 microgram/ml) activity was generally comparable to that of trovafloxacin (MIC90, 0.25 microgram/ml), or sparfloxacin (MIC90, 0.5 microgram/ ml) and markedly superior to ofloxacin (MIC90, 2-4 micrograms/ml) against the streptococci. Rates of penicillin non-susceptibility were 41.9, 38.0, and 16.2% for S. pneumoniae (301 strains), viridans group streptococci (150 strains), and beta-haemolytic streptococci (148 strains). Etest results correlated well (95.7-100.0% +/- one log2 dilution) with the reference MIC results, but Etest tended to have elevated gatifloxacin MIC results compared to the broth microdilution method for the highly resistant isolates (MICs, > 2 micrograms/ml). Gatifloxacin disk zone diameters correlate well to reference MICs for all streptococci and proposed interpretive criteria (susceptible at < or = 1 microgram/ml or > or = 18 mm, and resistant at > or = 4 micrograms/ml or < or = 14 mm) did not produce discords between method results (absolute agreement). A nine laboratory quality control (QC) study conforming to the National Committee for Clinical Laboratory Standards (NCCLS) Guideline M23-T3 studied S. pneumoniae ATCC 49619 and gatifloxacin. Proposed ranges for QC of NCCLS tests were 0.12-0.5 microgram/ml for the broth microdilution test and 24-31 mm for the disk diffusion method. These reported results indicate that gatifloxacin was a potent fluoroquinolone with extensive activity against streptococcal isolates. In vitro test methods to measure this activity appear accurate and comparable; and QC guidelines have been established for routine clinical laboratory use pending approval by the NCCLS and the Food and Drug Administration (FDA).

Antimicrobial activity of SCH27899 (Ziracin®), a novel everninomicin derivative, tested against Streptococcus spp.: disk diffusion/Etest method evaluations and quality control guidelines

To combat the increasing rates of penicillin resistance among pneumococci and viridans group streptococci, new Gram-positive active agents are needed to avoid the overuse of vancomycin. SCH27899 is an everninomicin derivative with strong activity against glycopeptide-resistant enterococci, oxacillin-resistant staphylococci, and penicillin-resistant streptococci. This study tests the in vitro activity of SCH27899 against 304 strains of streptococci and evaluates the quality of the agar dilution, broth microdilution, disk diffusion, and Etest methods for this antimicrobial agent. Quality-control (QC) ranges for SCH27899 are also proposed. SCH27899 broth microdilution MICs among the penicillin-susceptible and -resistant streptococci tested ranged from < or = 0.008-0.5 microgram/mL. Organism groups with their respective MIC90s were as follows: Streptococcus pneumoniae (100 strains) and beta-haemolytic streptococci (70 strains), 0.12 microgram/mL; Streptococcus bovis (10 strains), 0.25 microgram/mL; and viridans group streptococci (124 strains), 0.5 microgram/mL. Etest SCH27899 MICs correlated well with broth microdilution MICs (92% +/- one log2 dilution, 98% +/- two log2 dilutions). Agar dilution SCH27899 MICs correlated well with broth microdilution MICs, but a shift toward slightly higher agar dilution MICs was attributed to difficulties in reading trailing endpoints with this method. Three concentrations (2.5, 5, and 10 micrograms) of SCH27899 were used for the disk diffusion method with small inhibition zone diameters (range, 11 to 19 mm) and limited variation between diameters (+/- 2 mm) as a result, both products of this compounds high molecular weight and poor diffusion through agar mediums. Proposed control ranges for SCH27899 when testing S. pneumoniae ATCC 49619 from a nine-center (30 tests per center) quality-control trial are < or = 0.016 to 0.032 microgram/mL for Etest, and 0.008 to 0.032 microgram/mL for broth microdilution tests from an earlier study. Because of the limited diffusion ability and bacteriostatic nature of SCH27899, MICs should be read at 80% of inhibition with agar in vitro systems (Etest, agar dilution), and the disk diffusion method is not recommended.

Comparative antimicrobial activity of gatifloxacin tested against Campylobacter jejuni including fluoroquinolone-resistant clinical isolates

Campylobacter jejuni is an important pathogen that causes gastroenteritis, as well as other disease states such as meningitis and septic arthritis. In this study, the Etest (AB BIODISK, Solna, Sweden) results were compared to a reference agar dilution method using gatifloxacin, a new 8-methoxyfluoroquinolone. A total of 53 strains of C. jejuni initially isolated from patients in California and Mexico were tested. Results demonstrated a high correlation (r = 0.88) between the two utilized in vitro dilution methods. In addition, gatifloxacin activity was compared to that of ciprofloxacin, metronidazole, amoxicillin, erythromycin, chloramphenicol, gentamicin, tetracycline, and trimethoprim/sulfamethoxazole using the Etest. Gatifloxacin (MIC90, 4 micrograms/ml) was approximately eight- to 16-fold more potent than ciprofloxacin (Mic90, > 32 micrograms/ml), a commonly used fluoroquinolone for Campylobacter infections. Eight strains highly resistant to ciprofloxacin (MIC90, > 32 micrograms/ml) were tested for cross resistance against the newer fluoroquinolones (gatifloxacin, levofloxacin, trovafloxacin) and the rank order of potency was: gatifloxacin (MIC50, 16 micrograms/ml) > trovafloxacin = levofloxacin (MIC50, > 32 micrograms/mL). However, only 25% ciprofloxacin-resistant strains were inhibited by < or = 1 microgram/mL of gatifloxacin or trovafloxacin. These results for gatifloxacin against C. jejuni strains must be further assessed in the context of in vivo trials before the clinical role of this new fluoroquinolone can be determined. The Etest appears to be a simple and precise susceptibility test method for testing C. jejuni isolates against fluoroquinolones and other alternative therapeutic agents.

Evaluation of the etest for antimicrobial spectrum and potency determinations of anaerobes associated with bacterial vaginosis and peritonitis

One hundred ninety-seven anaerobic organisms (24 Gardnerella vaginalis, 16 Mobiluncus spp., 19 Peptostreptococcus spp., 20 Lactobacillus spp., 20 Prevotella bivia/disiens, 81 Bacteroides fragilis group, 12 Clostridium spp., and five Fusobacterium spp.) were processed by the Etest (AB Biodisk, Solna, Sweden) and a reference (Brucella blood agar) method against 10 antimicrobial agents. For the bacterial vaginosis-associated pathogens, the Etest was more reproducible and correlated acceptably with the reference agar test: within +/- 1 log2 dilution for 74.4% of Mobiluncus spp. to 96.0% for Peptostreptococcus spp. (all organisms, 83.4%). The quantitative correlation +/- 2 log2 dilution steps between test results was 94.3%. Results with B. fragilis group strains demonstrated 97.3% correlation (+/- 2 log2 dilution) with a trend toward slightly lower Etest minimum inhibitory concentrations for ampicillin-sulbactam, cefotaxime, imipenem, and clindamycin. The absolute qualitative interpretive agreement between Etest and the reference agar dilution method results was 94.4%, with only a 0.4% false-susceptible error rate. The Etest appears to be a very practical, quantitatively accurate, alternative procedure for clinical microbiology laboratories routinely testing the susceptibilities of anaerobes and, by these presented data, organisms associated with female tract infections.

Evaluation of gemifloxacin (SB-265805, LB20304a): in vitro activity against over 6000 gram-positive pathogens from diverse geographic areas.

Gemifloxacin (GEMI), formerly SB-265805 and LB20304, is a newer fluoroquinolone with broad-spectrum activity against a wide variety of bacterial pathogens. The present investigation extended earlier observations by sampling an additional 6790 gram-positive organisms from more than 50 medical centres on three continents. The reference broth microdilution method with recommended medium supplements was used throughout. Selected results (number strains tested; MIC90 for GEMI/trovafloxacin in mg/l; % < or = 1 mg/l for GEMI/trovafloxacin) were: Staphylococcus aureus (3672; 2/2; 86/85), S. epidermidis (404; 1/>4; 92/71), Enterococcus faecalis (630; 4/>4; 76/66), E. faecium (216; > 4/>4; 15/11), Streptococcus pneumoniae (300; 0.06/0.25; 100/97), beta-haemolytic streptococci (150; 0.06/0.25; 100/100) and viridans group streptococci (150; 0.12/0.25; 99/97). Gemifloxacin appeared equal or superior to trovafloxacin in its overall gram-positive spectrum of activity pending a choice of the susceptible breakpoint concentration. Continued in vitro, pharmacodynamic and clinical investigations of gemifloxacin appear warranted.

Avoparcin, a glycopeptide used in animal foods: Antimicrobial spectrum and potency tested against human isolates from the United States

Avoparcin is a glycopeptide antimicrobial that is widely used as a growth promoter in animal feeds in portions of Western Europe. Recently, concern has emerged about the possible contribution of avoparcin use to the emergence of glycopeptide resistance in enterococci. Relatively little information exists on the spectrum of activity and potency of avoparcin. We studied the activity of avoparcin compared to vancomycin, teicoplanin, and 3 other antimicrobials against 814 recent human clinical isolates, including Staphylococcus spp. (240 strains), Streptococcus spp. (90 strains), and Enterococcus spp. (60 strains), using reference susceptibility test methods. Our results indicate that avoparcin was less potent than either vancomycin or teichoplanin against staphylococci (MIC50, 4 micrograms/ mL). There was a good correlation of avoparcin MICs with the MICs for vancomycin and teichoplanin for most species; however, the avoparcin MICs for Enterococcus spp. of the vanB phenotype were quite variable (MIC range, 2 to > 16 micrograms/mL). For Staphylococcus haemolyticus, high avoparcin MICs (> or = 16 micrograms/mL) were associated with oxacillin resistance. These results are relevant to the debate concerning the appropriateness of continued use of avoparcin as a growth promoter in animal husbandry. In particular, avoparcin as a glycopeptide with limited potency against some staphylococci seems to represent a theoretically greater risk for selecting glycopeptide resistance among staphylococci, but may not represent any greater threat for the selection of resistance in enterococci.

Susceptibility testing interpretive criteria for levofloxacin when testing respiratory pathogens, Haemophilus influenzae and Moraxella catarrhalis

The more active L-isomer, levofloxacin, of the racemic ofloxacin mixture has been under development for therapeutic use. In this study, we evaluated the activity of ofloxacin, levofloxacin, and D-ofloxacin against the fastidious respiratory tract pathogens Haemophilus influenzae and Moraxella catarrhalis. Levofloxacin was two-fold more active than ofloxacin against H. influenzae (MIC90, 0.015 microgram/ml), and D-ofloxacin was least active (MIC90, 1 microgram/ml). For M. catarrhalis the MIC90 values were 0.03 microgram/ml, 0.06 microgram/ml, and 2 micrograms/ml for levofloxacin, ofloxacin, and D-ofloxacin, respectively. For disk diffusion susceptibility testing, Chocolate Mueller-Hinton agar (CMH) was considered preferable to Haemophilus test medium (HTM) because it supported the growth of all of 105 H. influenzae strains whereas five strains failed to grow on HTM. In addition, the margins of the zones of inhibition were more distinct on CMH and the Haemophilus species strains with elevated fluoroquinolone MICs were readily distinguished. The superior growth on CMH was reflected in a reduction of inhibition zone diameters of 2-3 mm relative to the inhibition zone diameters on HTM. The previously proposed interpretive criteria for the 5 microgram disk diffusion susceptibility test (susceptible at > or = 17 mm) results in complete categorical agreement with the reference microdilution broth method for M. catarrhalis on Mueller Hinton agar and for H. influenzae on HTM and CMH. However, the minimum diameter of the zone of inhibition recorded for a member of the dominant population of either species was considerably greater (25 mm) than 17 mm on any of the media tested.

Gatifloxacin (AM-1155, CG 5501) susceptibility testing interpretive criteria and quality control guidelines for dilution and disk (5-μg) diffusion methods

Gatifloxacin (formerly AM-1155 and CG5501), a new 8-methoxy fluoroquinolone, has an expanded spectrum of activity against Gram-positive cocci and some anaerobic bacteria. This compound was tested against 600 recent clinical strains of rapidly growing aerobic species to establish susceptibility testing interpretive criteria for the reference broth microdilution and standardized disk (5-microgram) diffusion methods of the National Committee for Clinical Laboratory Standards (NCCLS). These strains included 285 Enterobacteriaceae (17 species), 165 staphylococci, 49 enterococci, and 101 nonfermentative Gram-negative bacilli. Based on achievable serum levels with projected gatifloxacin dosing regimens, MIC break points of or = 18 mm) for susceptibility and > or = 8 micrograms/mL (< or = 14 mm) for resistance were selected. The absolute agreement between tests was 94.3% with no very major false-resistant errors. The quality control ranges (MIC and zone diameters) for the NCCLS recommended strains were determined in a nine-laboratory NCCLS protocol as follows: Escherichia coli ATCC 25922 = 0.008-0.03 microgram/mL and 31-37 mm; Enterococcus faecalis ATCC 29212 = 0.12-1 microgram/mL; Pseudomonas aeruginosa ATCC 27853 = 0.5-2 micrograms/ml and 21-27 mm; Staphylococcus aureus ATCC 25923 = 27-33 mm and S. aureus ATCC 29213 = 0.03-0.12 microgram/mL. Gatifloxacin appears to be a promising new fluoroquinolone with acceptable susceptibility testing methods for routine clinical laboratory use.

Development of in vitro susceptibility testing methods for gemifloxacin (formerly LB20304a or SB-265805), an investigational fluoronaphthyridone

Potent investigational fluoroquinolones require convenient, but accurate, diagnostic tests for initially applied clinical trials. For this purpose, gemifloxacin (formerly SB-265805, LB20304a) was tested by the reference dilution tests and standardized disk diffusion methods of the National Committee for Clinical Laboratory Standards (NCCLS) to establish interpretive criteria. For rapid-growing pathogens, 986 organisms were tested by broth microdilution MIC, and 5- and 10-microgram disk diffusion tests. Correlation (r) between 5- and 10-microgram disk zone diameters was 0.99 (y = -0.12 to 0.99x) and the preferred 5-microgram disk zone/MIC scattergram produced a regression of y = 14.8 to 0.41x (r = 0.93). At potential pharmacodynamics (Cmax = 1.3 micrograms/mL for 320 mg dose) validated breakpoints of < or = 0.5 microgram/mL for susceptible and > or = 2 micrograms/mL for resistant, correlate zones of > or = 17 mm and < or = 13 mm produced rare serious interpretive errors (0.1%) and 96.7% absolute categorical agreement. For 304 Streptococcus pneumoniae and 305 strains of other streptococci, the same breakpoints produced 100 and 99.1% categorical accuracy even when testing levofloxacin-resistant (MIC, > or = 4 micrograms/mL) strains. Interpretive breakpoints were proposed for Hemophilus influenzae (300 strains tested), with complete correlation between tests. Etest (AB BIODISK, Solna, Sweden) was compared in all experiments with the fastidious species and showed a trend toward higher values (twofold). Gemifloxacin in vitro susceptibility test methods seem to be accurate and with very acceptable intermethod agreement, supported by previously reported functional quality control guidelines.