Multicenter Investigation of Gepotidacin (GSK2140944) Agar Dilution Quality Control Determinations for Neisseria gonorrhoeae ATCC 49226.

Gepotidacin, a novel triazaacenaphthylene antibacterial agent, is the first in a new class of type IIA topoisomerase inhibitors with activity against many biothreat and conventional pathogens, including Neisseria gonorrhoeae To assist ongoing clinical studies of gepotidacin to treat gonorrhea, a multilaboratory quality assurance investigation determined the reference organism (N. gonorrhoeae ATCC 49226) quality control MIC range to be 0.25 to 1 µg/ml (88.8% of gepotidacin MIC results at the 0.5 µg/ml mode).

Linezolid Surveillance Results for the United States (LEADER Surveillance Program 2014).

Thelinezolidexperience andaccuratedetermination ofresistance (LEADER) surveillance program has monitored linezolid activity, spectrum, and resistance since 2004. In 2014, a total of 6,865 Gram-positive pathogens from 60 medical centers from 36 states were submitted. The organism groups evaluated wereStaphylococcus aureus(3,106), coagulase-negative staphylococci (CoNS; 797), enterococci (855),Streptococcus pneumoniae(874), viridans group streptococci (359), and beta-hemolytic streptococci (874). Susceptibility testing was performed by reference broth microdilution at the monitoring laboratory. Linezolid-resistant isolates were confirmed by repeat testing. PCR and sequencing were performed to detect mutations in 23S rRNA, L3, L4, and L22 proteins and acquired genes (cfrandoptrA). The MIC50/90forStaphylococcus aureuswas 1/1 µg/ml, with 47.2% of isolates being methicillin-resistantStaphylococcus aureus Linezolid was active against allStreptococcus pneumoniaestrains and beta-hemolytic streptococci with a MIC50/90of 1/1 µg/ml and against viridans group streptococci with a MIC50/90of 0.5/1 µg/ml. Among the linezolid-nonsusceptible MRSA strains, one strain harboredcfronly (MIC, 4 µg/ml), one harbored G2576T (MIC, 8 µg/ml), and one containedcfrand G2576T with L3 changes (MIC, ≥8 µg/ml). Among CoNS, 0.75% (six isolates) of all strains demonstrated linezolid MIC results of ≥4 µg/ml. Five of these were identified asStaphylococcus epidermidis, four of which containedcfrin addition to the presence of mutations in the ribosomal proteins L3 and L4, alone or in combination with 23S rRNA (G2576T) mutations. Six enterococci (0.7%) were linezolid nonsusceptible (≥4 µg/ml; five with G2576T mutations, including one with an additionalcfrgene, and one strain withoptrAonly). Linezolid demonstrated excellent activity and a sustained susceptibility rate of 99.78% overall.

Determination of Disk Diffusion and MIC Quality Control Guidelines for Solithromycin, a Novel Fluoroketolide Antibacterial, against Neisseria gonorrhoeae.

This solithromycin quality control study was performed to establish quality control (QC) ranges for the N. gonorrhoeae ATCC 49226 control strain for MIC agar dilution testing (AD) and zones by disk diffusion testing (DD). The following ranges were established: AD, 0.03 to 0.25 µg/ml, and DD, 33 to 43 mm. In January 2015, the CLSI Subcommittee on Antimicrobial Susceptibility Testing approved these ranges, which will be important when evaluating solithromycin against clinical isolates of N. gonorrhoeae.

In vitro activity of linezolid as assessed through the 2013 LEADER surveillance program.

The 2013 LEADER surveillance program monitored the in vitro activity of linezolid and comparator agents against Gram-positive bacteria at 60 medical centers in the United States. A total of 7183 pathogens were contributed from 6 predetermined pathogen groups. The groups were Staphylococcus aureus, coagulase-negative staphylococci, enterococci, Streptococcus pneumoniae, ß-hemolytic streptococci, and viridans group streptococci. The MIC90 value for each of the 6 pathogen groups was 1 µg/mL. Susceptibility of "all organisms" to linezolid was 99.83%. Only 12 isolates (2 S. aureus, 3 Staphylococcus epidermidis, 1 Streptococcus sanguinis, 5 Enterococcus faecium, and 1 Enterococcus faecalis) were nonsusceptible to linezolid (0.17%). Three of these (2 S. aureus and 1 E. faecium) harbored the cfr resistance mechanism. The findings indicate that linezolid activity remains stable, although there are examples of clonal dissemination within several monitored institutions.

Quality control MIC ranges used for telavancin with application of a revised CLSI reference broth microdilution method.

The telavancin broth microdilution susceptibility testing method was revised, which provides MIC results lower than those obtained by the previous method. This study was performed to reestablish the quality control ranges for telavancin when tested against the strains (updated MIC range) Staphylococcus aureus ATCC 29213 (0.03 to 0.12 µg/ml), Enterococcus faecalis ATCC 29212 (0.03 to 0.12 µg/ml), and Streptococcus pneumoniae ATCC 49619 (0.004 to 0.015 µg/ml).

Determination of disk diffusion and MIC quality control guidelines for GSK2140944, a novel bacterial type II topoisomerase inhibitor antimicrobial agent.

GSK2140944 is a novel bacterial type II topoisomerase inhibitor in development for the treatment of conventional and biothreat pathogens, including Gram-positive pathogens and methicillin-resistant Staphylococcus aureus. This quality control study was performed to establish ranges for selected control strains: S. aureus ATCC 29213 and ATCC 25923, Escherichia coli ATCC 25922, Haemophilus influenzae ATCC 49247, and Streptococcus pneumoniae ATCC 49619. The control ranges will be crucial for the accurate evaluation of GSK2140944 potency as it progresses through clinical trial development.

Summary of linezolid activity and resistance mechanisms detected during the 2012 LEADER surveillance program for the United States.

This study summarizes the linezolid susceptibility testing results for 7,429 Gram-positive pathogens from 60 U.S. sites collected during the 2012 sampling year for the LEADER Program. Linezolid showed potent activity when tested against 2,980 Staphylococcus aureus isolates, inhibiting all but 3 at ≤2 µg/ml. Similarly, linezolid showed coverage against 99.5% of enterococci, as well as for all streptococci tested. These results confirm a long record of linezolid activity against U.S. Gram-positive isolates since regulatory approval in 2000.

Streptococcus sanguinis isolate displaying a phenotype with cross-resistance to several rRNA-targeting agents.

This study describes a clinical case of a 71-year-old male with a history of ischemic cardiomyopathy after left ventricular assist device (LVAD) endocarditis caused by methicillin-resistant Staphylococcus epidermidis (MRSE) and a rare linezolid-resistant Streptococcus sanguinis strain (MIC, 32 µg/ml). The patient received courses of several antimicrobial agents, including linezolid for 79 days. The S. sanguinis strain had mutations in the 23S rRNA (T2211C, T2406C, G2576T, C2610T) and an amino acid substitution (N56D) in L22 and exhibited cross-resistance to ribosome-targeting agents.

Educational antimicrobial susceptibility testing as a critical component of microbiology laboratory proficiency programs: American Proficiency Institute results for 2007-2011.

External laboratory proficiency programs are an important requirement for test quality assurance (EQA) and compliance to regulatory guidelines (Clinical Laboratory Improvement Amendments and inspections). The American Proficiency Institute (API) regularly distributes EQA sample challenges (test events) including an Educational Sample (ES) for antimicrobial susceptibility testing. Beginning in 2007, API has sent 3 ES samples annually, each a well-characterized (molecular/phenotypic methods) strain having an interesting/emerging mechanism of resistance. Hundreds of USA laboratories, usually serving small- to medium-size hospitals and clinics, participate in the API ungraded ES test event. Analysis of responses is made and reported electronically as ES critiques addressing contemporary susceptibility testing issues that affect patient therapy. Seven Gram-positive (+) and 8 Gram-negative (-) ES strains were tested over the 5 years (2007-2011) with organism identification (graded) accuracy of 95.3% (range, 91.0-99.2%) for Gram (-) and 97.0% (range, 94.2-100.0%) for Gram (+) challenges. Susceptibility testing categorical accuracy was generally greatest for the disk diffusion test (91.0/97.0%) compared to the MIC methods (commercial automated or manual) combined (89.9/96.1%, for Gram [-]/Gram [+], respectively). The most worrisome observations of these ES samples were as follows: 1) poor recognition of ESBL- and serine carbapenemase-producing strains (various types including Klebsiella pneumoniae carbapanemase) due to delayed application of Clinical and Laboratory Standards Institute [CLSI] guidelines; 2) overcalling of ESBL in organisms having wild-type non-ESBL enzymes (OXA series; OXA, 1/30) due to commercial system or participant interpretive error; and 3) occasional drug-bug discords noted in nonfermentative Gram (-) bacilli. In conclusion, the API ES series of ungraded susceptibility testing challenges (accuracy was >90%) has been well received by subscribers and has provided detailed educational opportunities to improve laboratory testing performance. ES samples have delivered guidance to enable laboratories to rapidly comply with CLSI document changes of interpretive breakpoints such as those for ß-lactams when testing Enterobacteriaceae and Pseudomonas aeruginosa; the program was sustained into 2012 and beyond to document quality of susceptibility tests in USA laboratories.

An international activity and spectrum analysis of linezolid: ZAAPS Program results for 2011.

Through a continuing resistance surveillance monitoring program, linezolid was shown to maintain its spectrum and potency against a collection of 8059 clinically relevant Gram-positive strains collected from patients at 79 medical centers in 33 countries and Hong Kong. Linezolid MIC90 values were 2 µg/mL for methicillin-resistant and -susceptible Staphylococcus aureus and enterococci, and the MIC90 value was 1 µg/mL for coagulase-negative staphylococci (CoNS), ß-hemolytic streptococci, Streptococcus pneumoniae, and viridans group streptococci. Reference broth microdilution susceptibility testing for linezolid demonstrated a 99.83% susceptibility rate for all organisms. All S. aureus were inhibited by ≤2 µg/mL. Three (0.3%) of 928 strains of CoNS had a linezolid MIC of 4 µg/mL and contained the cfr resistance gene; 1 also had a mutation in L3. There were 14 linezolid-resistant strains detected from 7 countries (Brazil [5], France [1], Germany [2] Greece [2], Italy [2], Ireland [1], and Spain [1]) representing 5 species (E. faecium, S. capitis, S. epidermidis, S. hominis, S. lugdenensis). A mobile cfr gene was noted in 2 species having elevated linezolid MIC values; one was a S. haemolyticus isolate with a MIC at 4 µg/mL. Resistance rates were as follows for the 6 groups of organisms sampled in the 2011 ZAAPS Program: CoNS, 1.2%; enterococci, 0.39%; among S aureus, S. pneumoniae, viridans group streptococci, and ß-hemolytic streptococci, no resistance was detected. As the activities of commonly used antimicrobials continue to be compromised by evolving resistance mechanisms in Gram-positive pathogens, linezolid-resistant strains remain uncommon and without increasing occurrence.

Determination of disk diffusion and MIC quality control guidelines for GSK2251052: a novel boron-containing antibacterial.

GSK2251052 is a boron-containing antimicrobial agent in clinical development for the treatment of serious Gram-negative bacterial infections. These GSK2251052 quality control (QC) studies were performed to establish ranges for control strains (broth microdilution [BMD] MIC and disk diffusion zones) as follows: Pseudomonas aeruginosa ATCC 27853 (2-8 µg/mL and 15-24 mm), Escherichia coli ATCC 25922 (0.5-2 µg/mL and 23-30 mm), Haemophilus influenzae ATCC 49247 (0.25-1 µg/mL and 22-31 mm), Streptococcus pneumoniae ATCC 49619 (0.25-1 µg/mL and 19-28 mm), Bacteroides fragilis ATCC 25285 (1-4 µg/mL [BMD] and 1-4 µg/mL [agar dilution]), and Bacteroides thetaiotaomicron ATCC 29741 (1-8 µg/mL [BMD] and 2-8 µg/mL [agar dilution]). These ranges, approved by Clinical and Laboratory Standards Institute, will be crucial in accurately evaluating GSK2251052 in vitro potency.

Linezolid surveillance results for the United States: LEADER surveillance program 2011.

The LEADER surveillance program monitors the in vitro activity of linezolid and comparator agents against Gram-positive bacteria in the United States. In its eighth consecutive year (2011), a total of 60 medical centers from the United States, including seven medical centers specializing in children's health care contributed a total of 7,303 Gram-positive pathogens. The MIC(90) value for Staphylococcus aureus was 2 µg/ml, and for coagulase-negative staphylococci, enterococci, Streptococcus pneumoniae, ß-hemolytic streptococci, and viridans group streptococci, the MIC(90) was 1 µg/ml. The "all organism" linezolid-resistant and nonsusceptible rate was only 0.19%.

ZAAPS Program results for 2010: an activity and spectrum analysis of linezolid using clinical isolates from 75 medical centres in 24 countries.

The Zyvox® Annual Appraisal of Potency and Spectrum (ZAAPS) Program monitors the in vitro activities of linezolid and comparator agents for Gram-positive organisms in Latin America, Europe, Canada, and the Asia-Pacific. For the 2010 Program a total of 6305 Gram-positive strains were collected from 75 medical centres on five continents (24 countries). Reference broth microdilution susceptibility tests were performed on organisms from the following groups: Staphylococcus aureus (2875), coagulase negative staphylococci (CoNS) (855), enterococci (787), Streptococcus pneumoniae (926), viridans group and other streptococci (325), and beta-haemolytic streptococci (507). Linezolid demonstrated a 99.81% susceptibility rate among 6305 strains tested from 24 nations. Of the resistant isolates, four linezolid-resistant strains of enterococci (two each for Enterococcus faecalis and Enterococcus faecium) were found in four nations (China, Thailand, Germany, and Brazil). Eight CoNS (Staphylococcus epidermidis, Staphylococcus hominis) isolates were observed to be resistant to linezolid (MIC, ≥8 µg/ml). Two strains from Mexico were determined to be from an ongoing epidemic, and investigations showed that isolates from Italy and Brazil were also from circulating resistant clones discovered in earlier years. MRSA rates varied by region and between nations, as did resistances to other potential therapeutic agent options frequently listed for MRSA therapy such as clindamycin, fluoroquinolones and trimethoprim/sulfamethoxazole. In summary, the 2010 ZAAPS Program demonstrated that linezolid activity remains stable around the world with >99% susceptibility.

Disk diffusion and MIC quality control ranges for BC-3205 and BC-3781, two novel pleuromutilin antibiotics.

MIC and disk diffusion quality control (QC) ranges were established for two new pleuromutilin antimicrobials (BC-3205 and BC-3781) in an eight-laboratory study performed according to Clinical and Laboratory Standards Institute M23-A3 guidelines. Staphylococcus aureus ATCC 29213 and 25923, Streptococcus pneumoniae ATCC 49619, and Haemophilus influenzae ATCC 4927 strains were evaluated. The proposed QC ranges would aid clinical laboratories in testing these compounds following their development for treatment of respiratory and cutaneous infections.

LEADER surveillance program results for 2010: an activity and spectrum analysis of linezolid using 6801 clinical isolates from the United States (61 medical centers).

The LEADER program monitors the in vitro activity of linezolid and comparator agents across the United States using reference broth microdilution and supportive molecular susceptibility-based investigations. This report summarizes the data from the 2010 program, the seventh consecutive year. A total of 61 medical centers from the USA including 7 medical centers specializing in children's healthcare provided a total of 6801 Gram-positive pathogens. The medical centers represented all 9 US Bureau of Census geographic regions. The organisms tested by reference broth microdilution were 3105 Staphylococcus aureus, 944 coagulase-negative staphylococci (CoNS), 934 Enterococci, 803 Streptococcus pneumoniae, 604 ß-haemolytic streptococci, and 411 viridans group and other streptococci. The MIC(90) value for each of the above 6 targeted groups of organisms was 1 µg/mL. The "all organism" linezolid-resistant and nonsusceptible rate was 0.38%, which has been constant at 0.34% (2009) to 0.45% (2006) for the last 4 years. For Staphylococcus aureus, only 0.06% of the isolates were linezolid-resistant (MIC, ≥8 µg/mL); however, 2 additional methicillin-resistant Staphylococcus aureus had a cfr and a MIC of only 4 µg/mL. Resistance to linezolid was detected in 7 enterococci (0.75%) and 14 CoNS isolates (1.48%). This also represents a stable rate of resistance noted since the 2006 LEADER program report. Of note, for the first time in the 7 years of the Leader Program a linezolid-resistant Streptococcus pneumoniae was encountered. Overall, the results of the LEADER program demonstrate that linezolid maintains excellent in vitro activity against target Gram-positive pathogens across the USA. The LEADER program continues to provide valuable reference and molecular-level monitoring of linezolid activity.

Determination of disk diffusion and MIC quality control ranges for GSK1322322, a novel peptide deformylase inhibitor.

GSK1322322 is a novel peptide deformylase inhibitor in the early phase of development for treatment of complicated bacterial skin and skin structure infection and hospitalized community-acquired pneumonia. This quality control (QC) study was performed to establish broth microdilution and disk diffusion QC ranges for strains Staphylococcus aureus ATCC 29213 (MIC range, 1 to 4 µg/ml), Haemophilus influenzae ATCC 49247 (MIC and disk diffusion zone diameter ranges, 0.5 to 4 µg/ml and 20 to 28 mm, respectively), Streptococcus pneumoniae ATCC 49619 (MIC and disk diffusion zone diameter ranges, 0.12 to 0.5 µg/ml and 23 to 30 mm, respectively), and S. aureus ATCC 25923 (disk diffusion zone diameter range, 18 to 26 mm). These ranges are crucial for evaluating GSK1322322 potency as it progresses through clinical trials.

Determination of disk diffusion and MIC quality control guidelines for JNJ-Q2, a novel quinolone.

JNJ-Q2 is a novel fluorinated 4-quinolone in development for treatment of acute bacterial skin and skin structure infection and community-acquired bacterial pneumonia. This quality control (QC) study was performed to establish ranges for control strains: Staphylococcus aureus ATCC 29213 (0.004 to 0.015 µg/ml), Enterococcus faecalis ATCC 29212 (0.015 to 0.06 µg/ml), Pseudomonas aeruginosa ATCC 27853 (0.5 to 2 µg/ml and 17 to 23 mm), Escherichia coli ATCC 25922 (0.008 to 0.03 µg/ml and 30 to 36 mm), Haemophilus influenzae ATCC 49247 (0.002 to 0.015 µg/ml and 31 to 39 mm), Streptococcus pneumoniae ATCC 49619 (0.004 to 0.015 µg/ml and 28 to 35 mm), and S. aureus ATCC 25923 (32 to 38 mm). These ranges will be crucial in evaluating JNJ-Q2 potency as it progresses through clinical trial development.

LEADER Program results for 2009: an activity and spectrum analysis of linezolid using 6,414 clinical isolates from 56 medical centers in the United States.

The LEADER Program monitors the in vitro activity of linezolid in sampled U.S. medical centers using reference broth microdilution methods with supporting molecular investigations in a central laboratory design. This report summarizes data obtained in 2009, the 6th consecutive year of this longitudinal study. A total of 6,414 isolates from 56 medical centers in all nine Census regions across the United States participated in 2009. For the six leading species/groups, the following linezolid MIC(90) values were observed: Staphylococcus aureus, 2 µg/ml; coagulase-negative staphylococci (CoNS), 1 µg/ml; Enterococcus spp., 2 µg/ml; Streptococcus pneumoniae, 1 µg/ml; viridans group streptococci, 1 µg/ml; and beta-hemolytic streptococci, 1 µg/ml. Linezolid resistance was only 0.34% overall, with no evidence of significant increase in the LEADER Program since 2006. The predominant linezolid resistant mechanism found was a G2576T mutation in the 23S rRNA. L3/L4 riboprotein mutations were also found. The mobile multidrug-resistant cfr gene was found in four strains (two S. aureus strains and one strain each of S. epidermidis and S. capitis) from four different states, suggesting persistence but a lack of dissemination. Linezolid continues to exhibit excellent activity and spectrum, and this study documents the need for continued monitoring of emerging mechanisms of resistance over a wide geographic area.

Stability of linezolid activity in an era of mobile oxazolidinone resistance determinants: results from the 2009 Zyvox® Annual Appraisal of Potency and Spectrum program.

Linezolid is the only oxazolidinone agent approved for clinical use and has been administered to millions of patients over nearly a decade, becoming an important therapeutic alternative for infections caused by multidrug-resistant (MDR) Gram-positive pathogens. Resistance is due to mutations in 23S rRNA and the ribosomal proteins L3 and L4 and, more recently, the mobile cfr gene (causes resistance to several antimicrobial classes). Using reference broth microdilution methods in a central reference laboratory design, MIC values were obtained during the 2009 Zyvox® Annual Appraisal of Potency and Spectrum program (5754 sampled strains from 22 countries), and the following MIC(90) values were obtained for the leading pathogen or species groups: Staphylococcus aureus (2 µg/mL), coagulase-negative staphylococci (CoNS; 1 µg/mL), Enterococcus spp. (2 µg/mL), and 3 groups of streptococci (1 µg/mL) including Streptococcus pneumoniae. Linezolid-resistant strains (8 or 0.14% overall) that were not Staphylococcus aureus were observed in 5 nations and included CoNS (0.48%) having the mobile cfr gene. The results of this study demonstrate that linezolid continues to be effective in vitro against MDR pathogens, and the resistance rates appear stable.