Chris M. Pillar

Monitoring antibiotic resistance in ocular microorganisms: results from the Antibiotic Resistance Monitoring in Ocular micRorganisms (ARMOR) 2009 surveillance study.

PURPOSE To determine the antibacterial susceptibility profile of bacterial pathogens from ocular infections against relevant aminoglycoside, β-lactam, cephalosporin, chloramphenicol, fluoroquinolone, glycopeptide, lincosamide, and macrolide antibacterial agents. DESIGN Laboratory investigation. METHODS Isolates from patients with bacterial eye infections were collected prospectively by 34 institutions across the United States and were submitted to a central laboratory for inclusion in the Antibiotic Resistance Monitoring in Ocular micRorganisms (ARMOR) study. Minimum inhibitory concentrations were determined by microbroth dilution for 200 Staphylococcus aureus (S. aureus), 144 coagulase-negative staphylococci, 75 Streptococcus pneumoniae (S. pneumoniae), 73 Haemophilus influenzae (H. influenzae), and 100 Pseudomonas aeruginosa (P. aeruginosa) isolates. RESULTS A large proportion of S. aureus and coagulase-negative staphylococci isolates were resistant to oxacillin/methicillin, azithromycin, or fluoroquinolones; 46.5% of S. aureus, 58.3% of coagulase-negative staphylococci, 9.0% of P. aeruginosa, and 9.3% of pneumococcal isolates were nonsusceptible to 2 or more antibacterial drug classes. Only 2.7% of H. influenzae isolates were nonsusceptible to 1 of the agents tested. Methicillin-resistant staphylococci were statistically more likely (all P < .0038) also to be resistant to fluoroquinolones, aminoglycosides, and macrolides. CONCLUSIONS Resistance to 1 or more antibiotics is prevalent among ocular bacterial pathogens. Current resistance trends should be considered before initiating empiric treatment of common eye infections.

Longitudinal survey of carbapenem resistance and resistance mechanisms in Enterobacteriaceae and non-fermenters from the USA in 2007–09

BACKGROUND Antibiotic resistance is problematic in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii, and is often associated with serious infections. Carbapenems are often one of the few remaining therapeutic options, so it is important to monitor carbapenem activity against these pathogens and to identify resistance mechanisms. METHODS Carbapenem susceptibilities were determined for 14 359 Enterobacteriaceae, 3614 P. aeruginosa and 994 A. baumannii from the USA (2007-09). Klebsiella pneumoniae with doripenem MICs ≥2 mg/L (n = 88), and P. aeruginosa (n = 452), A. baumannii (n = 349) and other enterics (n = 13) with doripenem MICs ≥4 mg/L were screened for carbapenem resistance mechanisms. RESULTS Doripenem/meropenem and imipenem susceptibilities for Enterobacteriaceae were >99% and 89%, respectively. Doripenem susceptibility (2007-09) for P. aeruginosa was 87.4%-84.1%; comparable to meropenem and higher than imipenem. For A. baumannii, doripenem susceptibility (2007-09) was 63%-58.2%; lower than imipenem and meropenem. Resistant K. pneumoniae had KPC and lacked porins OmpK35/OmpK36. In 2009, 3.4% of all K. pneumoniae possessed KPC. Five other enterics and one P. aeruginosa possessed KPC. Resistance mechanisms in P. aeruginosa were loss of porin OprD (90%), efflux (55%) and elevated AmpC activity (25%). Acquired carbapenemases OXA-23/-24 were present in 48% of resistant A. baumannii. VIM metallo-β-lactamases were present in three P. aeruginosa and one A. baumannii isolates. CONCLUSIONS Doripenem and meropenem were more active than imipenem against Enterobacteriaceae and P. aeruginosa from the USA. Carbapenem resistance mechanisms included serine carbapenemases, elevated AmpC activity, efflux and porin deficiencies occurring mostly in P. aeruginosa. Metallo-β-lactamases were found in <0.1% of isolates.

Besifloxacin, a Novel Fluoroquinolone, Has Broad-Spectrum In Vitro Activity against Aerobic and Anaerobic Bacteria

ABSTRACT The antibacterial spectrum of besifloxacin, a novel fluoroquinolone recently approved for treatment of ocular infections, was studied using 2,690 clinical isolates representing 40 species. Overall, besifloxacin was the most potent agent tested against gram-positive pathogens and anaerobes and was generally equivalent to comparator fluoroquinolones in activity against most gram-negative pathogens. Besifloxacin demonstrated potent, broad-spectrum activity, which was particularly notable against gram-positive and gram-negative isolates that were resistant to other fluoroquinolones and classes of antibacterial agents.

Bactericidal activity of besifloxacin against staphylococci, Streptococcus pneumoniae and Haemophilus influenzae

Objectives Besifloxacin is a novel fluoroquinolone that was recently approved for topical treatment of bacterial conjunctivitis. The compound was shown to be active in vitro against a broad spectrum of bacteria, including isolates resistant to other antibacterials. Here, the bactericidal activity of besifloxacin was evaluated against the most common bacterial conjunctivitis pathogens. Methods MIC, MBC and time–kill experiments with besifloxacin and comparators were performed according to CLSI guidelines. Quinolone resistance-determining regions (QRDRs) were sequenced using standard PCR-based techniques. Results MIC and MBC data indicated that besifloxacin was the most potent fluoroquinolone tested against Staphylococcus aureus (n = 30), Staphylococcus epidermidis (n = 15) and Streptococcus pneumoniae (n = 35), while all fluoroquinolones were highly active against Haemophilus influenzae (n = 40). Besifloxacin MBC:MIC ratios were ≤4 for 97.5% of all isolates tested (n = 120). All fluoroquinolones tested, as well as tobramycin, were bactericidal, while azithromycin was bactericidal against S. pneumoniae and H. influenzae, but bacteriostatic against the staphylococci. Time–kill assays with all four species showed that besifloxacin caused ≥1000-fold killing within 2 h for 11 of 12 isolates. Only one isolate treated with moxifloxacin and three ciprofloxacin-treated isolates achieved the same level of bactericidal activity under the same conditions. Unlike the comparator fluoroquinolones, besifloxacin maintained a high potency and bactericidal activity even against strains that contained multiple mutations in the genes encoding DNA gyrase and topoisomerase IV. Conclusions Overall, besifloxacin demonstrated rapid bactericidal activity against the four major human pathogens tested here, including isolates that showed in vitro resistance to other fluoroquinolones, β-lactams, macrolides or aminoglycosides.

Enterococcal virulence--pathogenicity island of E. Faecalis.

Enterococcal species, though most commonly regarded as members of the microbial flora of the intestinal tract, have recently emerged as human pathogens of significant concern. The rapid spread of antibiotic resistance among enterococci, which has resulted in strains now being routinely isolated that are resistant to all bactericidal regimens, has prompted considerable interest in investigating the pathogenesis of enterococcal infection, with a view toward deriving new, information-based treatment strategies. This review summarizes major findings on the pathogenesis of enterococcal infection, fits them into a model for the dual lifestyle of enterococci as commensal and pathogen, and integrates into that model a recently discovered pathogenicity island of Enterococcus faecalis.

Multidrug resistance among Acinetobacter spp. in the USA and activity profile of key agents: results from CAPITAL Surveillance 2010

Multidrug resistance among Acinetobacter spp. leaves few effective antibiotic options for treatment. To monitor antibiotic resistance in Acinetobacter spp., the US CAPITAL 2010 Surveillance data were evaluated by patient demographics, specimen source, and hospital ward. Isolates (N=514) were collected from 65 sites across the USA and Puerto Rico. Isolates were centrally tested for susceptibility to carbapenems and key antimicrobials by broth microdilution. Colistin was the most effective agent tested, with 95% susceptibility. The overall susceptibility of Acinetobacter spp. was low (39% for piperacillin/tazobactam, 41% for levofloxacin, 45% for ceftazidime, 47-51% for the carbapenems, and 58% for tobramycin). Multidrug resistance (MDR), defined as resistance to ≥3 antimicrobial agent groups, was detected in 54% of the isolates. MDR isolates were most common among elderly patients (65%), lower respiratory tract isolates (62%), and inpatient/intensive care unit isolates (54-58%). These data update trends in the distribution and prevalence of the MDR phenotype in Acinetobacter spp.

Structure and DNA-binding properties of the cytolysin regulator CylR2 from Enterococcus faecalis

Enterococcus faecalis is one of the major causes for hospital‐acquired antibiotic‐resistant infections. It produces an exotoxin, called cytolysin, which is lethal for a wide range of Gram‐positive bacteria and is toxic to higher organisms. Recently, the regulation of the cytolysin operon was connected to autoinduction by a quorum‐sensing mechanism involving the CylR1/CylR2 two‐component regulatory system. We report here the crystal structure of CylR2 and its properties in solution as determined by heteronuclear NMR spectroscopy. The structure reveals a rigid dimer containing a helix–turn–helix DNA‐binding motif as part of a five‐helix bundle that is extended by an antiparallel β‐sheet. We show that CylR2 is a DNA‐binding protein that binds specifically to a 22 bp fragment of the cytolysin promoter region. NMR chemical shift perturbation experiments identify surfaces involved in DNA binding and are in agreement with a model for the CylR2/DNA complex that attributes binding specificity to a complex network of CylR2/DNA interactions. Our results propose a mechanism where repression is achieved by CylR2 obstruction of the promoter preventing biosynthesis of the cytolysin operon transcript.

In vitro time–kill experiments with besifloxacin, moxifloxacin and gatifloxacin in the absence and presence of benzalkonium chloride

OBJECTIVES To compare the bactericidal activity of besifloxacin, moxifloxacin and gatifloxacin and determine the contribution of the preservative benzalkonium chloride (BAK) to bactericidal activity. METHODS Time-kill experiments were performed against four species (n=12) with besifloxacin, moxifloxacin and gatifloxacin, in the presence or absence of BAK, at t=0, 5, 15, 30, 45, 60, 120 and 360 min, according to standard CLSI methods. RESULTS In the presence of BAK, bactericidal activity was observed within 5 min, regardless of the fluoroquinolone tested. The bactericidal activity of BAK was unaffected by the concurrent presence of besifloxacin and rapid killing (within 5 to 15 min) was not observed at BAK concentrations below 50 mg/L. However, when tested without BAK, besifloxacin was bactericidal in as little as 45 min, while moxifloxacin and gatifloxacin required at least 120 min; besifloxacin kill rates against fluoroquinolone-susceptible and -resistant strains were at least 2- to 4-fold faster than those of gatifloxacin or moxifloxacin. CONCLUSIONS Besifloxacin was the most rapidly bactericidal fluoroquinolone tested, followed by gatifloxacin and moxifloxacin, both of which had similar activity. Our studies demonstrate that the previously reported rapid in vitro killing by gatifloxacin formulations was probably due to the concurrent presence of 50 mg/L BAK, which is much higher than the 3.2 mg/L BAK observed in human tears 1 min after instillation of ophthalmic gatifloxacin solutions [Friedlaender MH, Breshears D, Amoozgar B et al. The dilution of benzalkonium chloride (BAK) in the tear film. Adv Ther 2006; 23: 835-41].

Comparative in vitro activity of oritavancin and other agents against methicillin-susceptible and methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) infections constitute a threat to the public health due to their prevalence and associated mortality and morbidity. Several agents have been recently approved to treat MRSA skin infections including lipoglycopeptides (dalbavancin, oritavancin, and telavancin), ceftaroline, and tedizolid. This study compared the MIC, minimum bactericidal concentration (MBC), and time-kill of these agents alongside daptomycin, linezolid, and vancomycin against MRSA (n=15); meropenem, cefazolin, and nafcillin were also included against methicillin-susceptible S. aureus (MSSA [n=12]). MIC and MBC testing was conducted in accordance with Clinical and Laboratory Standards Institute guidelines, and time-kills were evaluated at multiples of the MIC and the free-drug maximum plasma concentration (fCmax) at both standard and high inoculum densities for a subset of MRSA (n=2) and MSSA (n=2). MRSA and MSSA were highly susceptible to all agents, with the lipoglycopeptides having the most potent activity by MIC50/90. All agents excluding tedizolid and linezolid were bactericidal by MBC for MRSA and MSSA, though dalbavancin and telavancin exhibited strain-specific bactericidal activity for MRSA. All agents excluding tedizolid and linezolid were bactericidal by time-kill at their respective fCmax against MRSA and MSSA at standard inoculum density, though oritavancin exhibited the most rapid bactericidal activity. Oritavancin and daptomycin at their respective fCmax maintained similar kill curves at high inoculum density. In contrast, the killing observed with other agents was typically reduced or slowed at high inoculum density. These data demonstrate the rapid bactericidal activity of oritavancin and daptomycin against S. aureus relative to other MRSA agents regardless of bacterial burden.

Comparative in vitro activity of oritavancin and other agents against vancomycin-susceptible and -resistant enterococci

Sir, Antibiotic research has focused on discovering agents with activity against MDR pathogens (e.g. ESKAPE pathogens), including VRE, which along with Staphylococcus aureus are commonly isolated from healthcare-associated infections. Several new agents have been approved for the treatment of skin and skin structure infections caused by MRSA and enterococci, including oxazolidinones (linezolid and tedizolid), lipoglycopeptides (oritavancin, dalbavancin and telavancin), a cyclic lipopeptide (daptomycin), a glycylcycline (tigecycline) and an anti-MRSA cephalosporin (ceftaroline). Lipoglycopeptides, though active against vancomycin-susceptible enterococci (VSE), have variable activity against VRE, with oritavancin being the sole agent maintaining potent activity against VanA-type VRE. We report here a direct comparison of the in vitro activity of lipoglycopeptides and other skin agents against vancomycinsusceptible and -resistant Enterococcus faecalis (VSEfa and VREfa) and Enterococcus faecium (VSEfm and VREfm). Comparative evaluations included MIC and MBC determinations and time–kill kinetics. Since variation in inoculum density has been shown to impact the activity of several of these agents against S. aureus, time–kill kinetics were assessed at both standard and high inoculum densities. The evaluated isolates consisted of 74 random non-duplicate clinical isolates of VSEfa, VREfa, VSEfm and VREfm from the Micromyx repository (Kalamazoo, MI, USA) and The Medicines Company (Ville Saint Laurent, Quebec, Canada). VanA-phenotype (vancomycin and teicoplanin resistant) and VanB-phenotype (vancomycin resistant and teicoplanin susceptible) VRE were selected based on prior glycopeptide susceptibility test history. Agents were handled per CLSI (formerly NCCLS) guidelines and had results within CLSI quality control ranges during testing. Evaluations of lipoglycopeptides incorporated polysorbate 80 at a final concentration of 0.002% (v/v). MIC and MBC values were determined in accordance with standard CLSI methods. The time–kill kinetics of select isolates (one per phenotype evaluated) at standard inoculum ( 5 10 cfu/ mL) and high inoculum ( 5 10 cfu/mL) were determined as described by Arhin et al. using a method derived from the CLSI for agents at their fCmax (calculated from the respective prescribing information as 16 mg/L for oritavancin, dalbavancin, linezolid, vancomycin and ceftaroline, 8 mg/L for telavancin, 4 mg/L for daptomycin and 1 mg/L for tedizolid). The activity of the tested agents against enterococci is summarized by species and phenotype in Table 1. Among E. faecalis, VSEfa were susceptible to all agents with the lipoglycopeptides having the most potent activity by MIC90. Ceftaroline and oritavancin were the only consistently bactericidal agents against VSEfa based on the proportion of isolates with MBC:MIC ratios of 4. Based on MIC90, oritavancin, daptomycin, linezolid, tedizolid and ceftaroline maintained potent activity against VanA VREfa. Ceftaroline maintained bactericidal activity against VanA VREfa, while the other agents typically had MBC:MIC ratios>4. Against VSEfm and VREfm (VanA and VanB phenotypes), oritavancin was the most potent agent evaluated based on MIC90. All agents, excluding ceftaroline, which was largely inactive against E. faecium, had potent activity against VSEfm by MIC90. Oritavancin, daptomycin, linezolid and tedizolid maintained potent activity against VREfm. Oritavancin was 16-fold more potent by MIC90 than the comparator lipoglycopeptides against VREfm. Against VSEfm, oritavancin and daptomycin were the only consistently bactericidal agents based on the proportion of isolates with MBC:MIC ratios of 4. Daptomycin maintained bactericidal activity by MBC:MIC ratio against VanA and VanB VREfm. Consistent trends in bactericidal activity were apparent for each agent by time–kill at fCmax across the evaluated E. faecalis (one isolate each of VSEfa and VanA VREfa) and E. faecium (one isolate each of VSEfm, VanA VREfm and VanB VREfm) isolates. At the standard inoculum density, oritavancin and daptomycin were rapidly bactericidal with 3 log killing typically achieved within 0.25 and 4 h, respectively, with singular exceptions (4 h for oritavancin and 1 h for daptomycin against the VanA VREfa isolate). Telavancin was typically bactericidal, with 3 log killing observed at 24 h at the standard inoculum density with the exception of the VanB VREfm isolate, as was ceftaroline for E. faecalis but not E. faecium. Vancomycin, dalbavancin, tedizolid and linezolid did not achieve 3 log killing at the standard inoculum density for any of the evaluated E. faecalis or E. faecium isolates. Only oritavancin maintained bactericidal activity at the high inoculum density, with similar kill kinetics to those observed at the standard inoculum density. Few available agents exist for treating VRE infections despite their prevalence and associated mortality and morbidity. Among agents with activity against enterococci, oritavancin, daptomycin, linezolid and tedizolid maintained potent activity against VanA/ VanB VRE isolates. Both daptomycin and oritavancin exhibited rapid bactericidal activity at fCmax against VSE and VRE at standard inoculum densities, but only oritavancin maintained rapid