Sulopenem Disk Development, Quality Control Range, and MIC to Disk Result Correlation for Enterobacterales Isolates.

Sulopenem disk masses of 2, 5, 10, and 20 µg were evaluated by susceptibility testing isolates by broth microdilution and disk diffusion. A 2-µg disk was chosen, and error-rate bounding analysis in accordance with Clinical and Laboratory Standards Institute (CLSI) guideline M23 was conducted using a proposed sulopenem susceptible/intermediate/resistant (S/I/R) interpretive criterion of ≤0.5/1/≥2 µg/mL. Among the evaluated Enterobacterales (n = 2,856), very few interpretive errors were observed (no very major errors and only one major error). An eight-laboratory quality control (QC) study was performed using the 2-µg disk, and 99.0% (470/475) of results were within a 7-mm range of 24 to 30 mm. Results were similar by disk lot and media, and no outlier sites were observed. A sulopenem 2-µg disk QC range for Escherichia coli 29522 of 24 to 30 mm was established by the CLSI. A 2-µg sulopenem disk performs accurately and reproducibly for testing of Enterobacterales.

Analysis of the effect of urine on the in vitro activity of gepotidacin and levofloxacin against Escherichia coli, Staphylococcus epidermidis, and Staphylococcus saprophyticus.

Gepotidacin is a novel agent in development for treatment of gonorrhea and uncomplicated urinary tract infection. This study determined the effect of urine on the in vitro activity of gepotidacin and levofloxacin against relevant bacteria. Study strains were tested by Clinical and Laboratory Standards Institute broth microdilution and with method variations: CAMHB with 25%, 50%, 100% urine and pH adjusted 100% urine. Mean dilution difference (DD) of urine minimum inhibitory concentration (MICs) were <1 dilution of CAMHB MICs with some exceptions: Gepotidacin mean DD: Escherichia coli and Staphylococcus saprophyticus 100% urine (1.5 and 1.2, respectively) and S. saprophyticus pH 7.3 and 8.1 adjusted 100% urine (1.5 and 1.4, respectively); Levofloxacin mean DD: S. saprophyticus pH 7.3 adjusted 100% urine (1.5) and all species pH 8.1 adjusted 100% urine (1.2-1.8). Effects of urine on gepotidacin and levofloxacin MICs was minimal and not inclusive of all strains. Further analysis is warranted to fully assess the impact of urine on gepotidacin activity.

Analysis of antimicrobial susceptibility testing methods and variables and in vitro activity of gepotidacin against urogenital Neisseria gonorrhoeae in men.

Gepotidacin is a triazaacenaphthylene antibiotic with activity against Neisseria gonorrhoeae including strains resistant to current agents. We tested 145 N. gonorrhoeae isolates by agar dilution according to Gonococcal Isolate Surveillance Program and Clinical and Laboratory Standards Institute methodologies. Gepotidacin demonstrated a minimum inhibitory concentration (MIC)50 of 0.25 µg/mL and a MIC90 of 0.5 µg/mL (highest gepotidacin MIC was 1 µg/mL) against the 145 N. gonorrhoeae isolates tested. We also assessed the impact of test variables on antimicrobial susceptibility test results for gepotidacin, ciprofloxacin, and ceftriaxone against 10 N. gonorrhoeae isolates. Media type had the biggest effect but wasn't specific to gepotidacin. Gepotidacin MICs were also affected by inoculum, pH, and 10% CO2. These in vitro data indicate that further study of gepotidacin is warranted for potential use in treating gonorrhea infections and highlight the importance of controlling for media type, inoculum, CO2, and pH when performing MIC testing with gepotidacin.

1,3-Dioxane-Linked Bacterial Topoisomerase Inhibitors with Enhanced Antibacterial Activity and Reduced hERG Inhibition.

The development of new therapies to treat methicillin-resistant Staphylococcus aureus (MRSA) is needed to counteract the significant threat that MRSA presents to human health. Novel inhibitors of DNA gyrase and topoisomerase IV (TopoIV) constitute one highly promising approach, but continued optimization is required to realize the full potential of this class of antibiotics. Herein, we report further studies on a series of dioxane-linked derivatives, demonstrating improved antistaphylococcal activity and reduced hERG inhibition. A subseries of analogues also possesses enhanced inhibition of the secondary target, TopoIV.

CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests.

Effective evaluations of antimicrobial susceptibility tests (ASTs) require robust study design. The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing has recognized that many published studies reporting the performance of commercial ASTs (cASTs) suffer from major design and/or analysis flaws, rendering the results difficult or impossible to interpret. This minireview outlines the current consensus of the Methods Development and Standardization Working Group of the CLSI Subcommittee on Antimicrobial Susceptibility Testing regarding best practices for systematic evaluation of the performance of an AST, including the analysis and presentation of essential data intended for publication.

Changes in the antibiotic susceptibility of anaerobic bacteria from 2007-2009 to 2010-2012 based on the CLSI methodology.

Antimicrobial susceptibility testing of anaerobic isolates was conducted at four independent sites from 2010 to 2012 and compared to results from three sites during the period of 2007-2009. This data comparison shows significant changes in antimicrobial resistance in some anaerobic groups. Therefore, we continue to recommend institutions regularly perform susceptibility testing when anaerobes are cultured from pertinent sites. Annual generation of an institutional-specific antibiogram is recommended for tracking of resistance trends over time.

Characterisation of carbapenem-resistant Enterobacteriaceae from the southwestern Ohio, northern Kentucky and southeastern Indiana region.

Although carbapenem-resistant Enterobacteriaceae (CRE) have been recognised worldwide, there are important gaps in regional prevalence data. This study sought to determine the occurrence and type of CRE in the Cincinnati tri-state region. Clinical microbiology laboratories in the region screened patient stool and rectal swab samples using chromogenic screening agar. Isolates of Enterobacteriaceae resistant to carbapenems were characterised using molecular methods. Area institutions screened 1939 patient samples. Of 18 Enterobacteriaceae that grew on the screening agar, 8 isolates were resistant to one or all carbapenems and 6 isolates (all Klebsiella spp.) tested positive for a blaKPC gene. This study provides guidance on the prevalence of KPC and the genetic characteristics of carbapenem-resistant Klebsiella pneumoniae isolates in the Cincinnati tri-state area. Additional similar local epidemiology studies are warranted to provide an understanding and control of the spread of CRE.

Multi-center and multi-method evaluation of in vitro activities of ceftaroline against S. aureus.

This five-site study was performed to assess the reproducibility of ceftaroline MIC and disk results for Staphylococcus aureus. Three commercial broth microdilution, three gradient diffusion and ceftaroline 5µg disk diffusion methods were compared to a reference broth microdilution method against challenge isolates (n = 41) and isolates collected at four European sites (n = 30/site). For four MIC methods (Sensititre and three gradient diffusion methods), 99.0% of consolidated MIC results were within +/- 1 dilution of the reference MIC. Categorical agreement rates based on EUCAST breakpoints for the challenge isolates were 75.6-100% and for disk testing were 78.0-92.7%. There was no clear distinction between isolates with MIC results of 1 and 2mg/L with regard to variation in MIC or molecular genotyping results. The addition of an intermediate category for isolates with MIC results of 2mg/L would help to identify these isolates as borderline susceptible/non-susceptible isolates.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically.

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, ß-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

Evidence for daptomycin Etest lot-related MIC elevations for Staphylococcus aureus.

MIC testing was performed simultaneously by Etest and broth microdilution (BMD) on 587 Staphylococcus aureus isolates submitted by local laboratories to a reference laboratory for confirmatory testing (May 2005 to July 2008). Testing bias was assessed for Etest to BMD MIC ratios. Categoric and essential agreement, very major (BMD nonsusceptible, Etest susceptible), and major (BMD susceptible, Etest nonsusceptible) errors were evaluated. Agar and broth calcium concentrations were consistent with current Clinical and Laboratory Standards Institute and manufacturer recommendations. There was a consistent bias for higher Etest MIC values compared with BMD. Ratios ranged from 0.25 to 4 (average 1.3), with substantial variability noted among the 8 different Etest lots tested. Overall, 6% of all ratios were >2.0. Categoric agreement and essential agreement among the 8 Etest lots ranged from 73% to 96% and 74% to 100%, respectively; very major errors ranged from 3% to 9%, and major errors ranged from 6% to 35%. However, most of the discrepancies were limited to 3 Etest lots.

Activity of levofloxacin alone and in combination with a DnaK inhibitor against gram-negative rods, including levofloxacin-resistant strains.

Synergy time-kill testing of levofloxacin alone and in combination with CHP-105, a representative DnaK inhibitor, against 50 gram-negative rods demonstrated that 34 of the 50 strains tested showed significant synergy between levofloxacin and CHP-105 after 12 h and 24 h. Fourteen of these 34 organisms were quinolone resistant (levofloxacin MICs of > or =4 microg/ml).

Interpretive categorical accuracy of fluoroquinolone reference broth microdilution MIC results when testing Streptococcus pneumoniae: selection of a surrogate testing agent.

The categorical agreement among MIC results for the fluoroquinolones tested (levofloxacin, moxifloxacin, gatifloxacin and gemifloxacin) was high (99.16-99.85%), and error rates were nil or very low when 1 compound was used as a surrogate for predicting susceptibility (not resistance) to another agent in the class. No error was observed when levofloxacin was selected as the group surrogate for pneumococcal testing.

Determination of moxifloxacin anaerobic susceptibility breakpoints according to the Clinical and Laboratory Standards Institute guidelines.

A summary of the key data presented to Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical and Laboratory Standards) in determination of moxifloxacin anaerobic breakpoints is presented. The breakpoint analysis required review of a variety of data, including bacteriologic and clinical outcomes by MIC of anaerobic isolates from prospective clinical trials in patients with complicated intra-abdominal infections, human and animal pharmacokinetic/pharmacodynamic (PK/PD) information and in vitro models, MIC distributions of indicated organisms, and animal model efficacy data for strains with MIC values around prospective breakpoints. The compilation of the various components of this breakpoint analysis supports the US Food and Drug Administration (FDA) and CLSI moxifloxacin anaerobic breakpoints of < or =2 mg/L (susceptible), 4 mg/L (intermediate), and > or =8 mg/L (resistant), and provides information to European investigators for interpretation of MICs prior to establishment of the European Committee on Antimicrobial Susceptibility Testing breakpoints.

Factors influencing broth microdilution antimicrobial susceptibility test results for dalbavancin, a new glycopeptide agent.

Performance of antimicrobial susceptibility tests with new agents requires careful consideration of the properties of the antimicrobial to ensure that the tests are standardized, reproducible, and reflect the true potency of the drug. Dalbavancin is a new glycopeptide with potent activity against gram-positive bacterial species. The investigations described here demonstrated that methodologic modifications of procedures are necessary to ensure consistent test results, both for quality control and for routine testing of clinical isolates. Dimethyl sulfoxide is the preferred primary solvent. The addition of 0.002% polysorbate-80 (a surfactant) to dalbavancin-containing wells in the reference broth microdilution assay resulted in consistent and reproducible MIC results for three quality control strains: Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Streptococcus pneumoniae ATCC 49619. The same degree of consistency was observed among clinical isolates of gram-positive bacterial species tested in several clinical laboratories. These results indicate that the addition of 0.002% (final concentration) of the surfactant in broth microdilution tests produces optimal dalbavancin MICs required for accurate and reproducible clinical laboratory tests, without untoward influences of substrate binding or media constituents.

Characterization of a daptomycin-nonsusceptible vancomycin-intermediate Staphylococcus aureus strain in a patient with endocarditis.

We analyzed the emergence of daptomycin nonsusceptibility in a patient with persistent vancomycin-intermediate Staphylococcus aureus (VISA) bacteremia. The daptomycin-nonsusceptible VISA's cell wall demonstrated a reduction in muramic acid O-acetylation, a phenotypic parameter not previously reported for VISA; some isolates also contained a single point mutation in the mprF gene.

Clinical progression of methicillin-resistant Staphylococcus aureus vertebral osteomyelitis associated with reduced susceptibility to daptomycin.

Daptomycin, a novel cyclic lipopeptide antibiotic, exhibits rapid bactericidal activity in vitro against most clinically relevant gram-positive organisms, including drug-resistant pathogens. Herein we describe a patient in whom methicillin-resistant Staphylococcus aureus with reduced susceptibility to daptomycin was responsible for bacteremia and progressive vertebral osteomyelitis during daptomycin therapy.

Evolving concepts of pharmaceutical company-sponsored surveillance studies.

As a result of increasing bacterial resistance to antimicrobial agents, there is a need to conduct studies that monitor changes in susceptibility. In addition to studying the emergence and dissemination of antibacterial resistance, pharmaceutical companies perform surveillance studies for a number of reasons. As an example, the Alexander Project was conducted to study community-acquired respiratory infections internationally over 10 years. The project's findings have been valuable in the study of antimicrobial resistance. The Alexander Project has also been instrumental in the study of the evolution of resistance genes and in predictions of future rates of resistance, as well as in establishing the importance of high-quality data, the complexity of the evolution of resistance, and the need to disseminate the results in a variety of formats. Although there has been a reduction in pharmaceutical company studies, consolidated efforts between industry, government, and private groups have increased. Future surveillance efforts by pharmaceutical companies will likely be more targeted and disease directed.

Comparative in vitro activity of a pharmacokinetically enhanced oral formulation of amoxicillin/clavulanic acid (2000/125 mg twice daily) against 9172 respiratory isolates collected worldwide in 2000.

OBJECTIVES: A new, pharmacokinetically enhanced, oral formulation of amoxicillin/clavulanic acid has been developed to overcome resistance in the major bacterial respiratory pathogen Streptococcus pneumoniae, while maintaining excellent activity against Haemophilus influenzae and Moraxella catarrhalis, including beta-lactamase producing strains. This study was conducted to provide in vitro susceptibility data for amoxicillin/clavulanic acid and 16 comparator agents against the key respiratory tract pathogens. METHODS: Susceptibility testing was performed on 9172 isolates collected from 95 centers in North America, Europe, Australia, and Hong Kong by broth microdilution MIC determination, according to NCCLS methods, using amoxicillin/clavulanic acid and 16 comparator antimicrobial agents. Results were interpreted according to NCCLS breakpoints and pharmacokinetic/pharmacodynamic (PK/PD) breakpoints based on oral dosing regimens. RESULTS: Overall, 93.5% of Streptococcus pneumoniae isolates were susceptible to amoxicillin/clavulanic acid at the current susceptible breakpoint of < or =2 microg/mL and 97.3% at the PK/PD susceptible breakpoint of < or =4 microg/mL for the extended release formulation. Proportions of isolates that were penicillin intermediate and resistant were 13% and 16.5%, respectively, while 25% were macrolide resistant and 21.8% trimethoprim/sulfamethoxazole resistant. 21.9% of Haemophilus influenzae were beta-lactamase producers and 16.8% trimethoprim/sulfamethoxazole resistant, >99% of isolates were susceptible to amoxicillin/clavulanic acid, cefixime, ciprofloxacin and levofloxacin at NCCLS breakpoints. The most active agents against Moraxella catarrhalis were amoxicillin/clavulanic acid, macrolides, cefixime, fluoroquinolones, and doxycycline. Overall, 13% of Streptococcus pyogenes were resistant to macrolides. CONCLUSION: The extended release formulation of amoxicillin/clavulanic acid has potential for empiric use against many respiratory tract infections worldwide due to its activity against species resistant to many agents currently in use.