A. Simon Lynch

Use of an Efflux-Deficient Streptococcus pneumoniae Strain Panel To Identify ABC-Class Multidrug Transporters Involved in Intrinsic Resistance to Antimicrobial Agents

ABSTRACT Thirteen derivatives of the Streptococcus pneumoniae TIGR4 strain in which putative drug efflux pumps were genetically inactivated were constructed and characterized. The results indicate that two linked genes encoding the ABC-type transporters SP2073 and SP2075 function together to confer intrinsic resistance to a series of structurally unrelated compounds, including certain fluoroquinolones.

In vitro evaluation of CBR-2092, a novel rifamycin-quinolone hybrid antibiotic: studies of the mode of action in Staphylococcus aureus.

ABSTRACT Rifamycins have proven efficacy in the treatment of persistent bacterial infections. However, the frequency with which bacteria develop resistance to rifamycin agents restricts their clinical use to antibiotic combination regimens. In a program directed toward the synthesis of rifamycins with a lower propensity to elicit resistance development, a series of compounds were prepared that covalently combine rifamycin and quinolone pharmacophores to form stable hybrid antibacterial agents. We describe mode-of-action studies with Staphylococcus aureus of CBR-2092, a novel hybrid that combines the rifamycin SV and 4H-4-oxo-quinolizine pharmacophores. In biochemical studies, CBR-2092 exhibited rifampin-like potency as an inhibitor of RNA polymerase, was an equipotent (balanced) inhibitor of DNA gyrase and DNA topoisomerase IV, and retained activity against a prevalent quinolone-resistant variant. Macromolecular biosynthesis studies confirmed that CBR-2092 has rifampin-like effects on RNA synthesis in rifampin-susceptible strains and quinolone-like effects on DNA synthesis in rifampin-resistant strains. Studies of mutant strains that exhibited reduced susceptibility to CBR-2092 further substantiated RNA polymerase as the primary cellular target of CBR-2092, with DNA gyrase and DNA topoisomerase IV being secondary and tertiary targets, respectively, in strains exhibiting preexisting rifampin resistance. In contrast to quinolone comparator agents, no strains with altered susceptibility to CBR-2092 were found to exhibit changes consistent with altered efflux properties. The combined data indicate that CBR-2092 may have potential utility in monotherapy for the treatment of persistent S. aureus infections.

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.

In Vitro Evaluation of CBR-2092, a Novel Rifamycin-Quinolone Hybrid Antibiotic: Microbiology Profiling Studies with Staphylococci and Streptococci

ABSTRACT We present data from antimicrobial assays performed in vitro that pertain to the potential clinical utility of a novel rifamycin-quinolone hybrid antibiotic, CBR-2092, for the treatment of infections mediated by gram-positive cocci. The MIC90s for CBR-2092 against 300 clinical isolates of staphylococci and streptococci ranged from 0.008 to 0.5 μg/ml. Against Staphylococcus aureus, CBR-2092 exhibited prolonged postantibiotic effects (PAEs) and sub-MIC effects (SMEs), with values of 3.2, 6.5, and >8.5 h determined for the PAE (3× MIC), SME (0.12× MIC), and PAE-SME (3× MIC/0.12× MIC) periods, respectively. Studies of genetically defined mutants of S. aureus indicate that CBR-2092 is not a substrate for the NorA or MepA efflux pumps. In minimal bactericidal concentration and time-kill studies, CBR-2092 exhibited bactericidal activity against staphylococci that was retained against rifampin- or intermediate quinolone-resistant strains, with apparent paradoxical cidal characteristics against rifampin-resistant strains. In spontaneous resistance studies, CBR-2092 exhibited activity consistent with balanced contributions from its composite pharmacophores, with a mutant prevention concentration of 0.12 μg/ml and a resistance frequency of <10−12 determined at 1 μg/ml in agar for S. aureus. Similarly, CBR-2092 suppressed the emergence of preexisting rifamycin resistance in time-kill studies undertaken at a high cell density. In studies of the intracellular killing of S. aureus, CBR-2092 exhibited prolonged bactericidal activity that was superior to the activities of moxifloxacin, rifampin, and a cocktail of moxifloxacin and rifampin. Overall, CBR-2092 exhibited promising activity in a range of antimicrobial assays performed in vitro that pertain to properties relevant to the effective treatment of serious infections mediated by gram-positive cocci.

Antibody-Based Biologics and Their Promise to Combat Staphylococcus aureus Infections

The growing incidence of serious infections mediated by methicillin-resistant Staphylococcus aureus (MRSA) strains poses a significant risk to public health. This risk is exacerbated by a prolonged void in the discovery and development of truly novel antibiotics and the absence of a vaccine. These gaps have created renewed interest in the use of biologics in the prevention and treatment of serious staphylococcal infections. In this review, we focus on efforts towards the discovery and development of antibody-based biologic agents and their potential as clinical agents in the management of serious S. aureus infections. Recent promising data for monoclonal antibodies (mAbs) targeting anthrax and Ebola highlight the potential of antibody-based biologics as therapeutic agents for serious infections.

Activities of carbapenem and comparator agents against contemporary US Pseudomonas aeruginosa isolates from the CAPITAL surveillance program

Antimicrobial susceptibilities of contemporary Pseudomonas aeruginosa clinical isolates were determined from the CAPITAL 2010 surveillance program. Isolates were collected from 100 sites throughout the USA and Puerto Rico, and included isolates representing a range of patient demographics and infection types. A total of 2722 isolates were tested for susceptibility to a broad spectrum of agents, with susceptibilities ranging from 98.8% for colistin to 74% for levofloxacin. Doripenem was the most active carbapenem agent, with 88.6% of isolates susceptible, in comparison with 78.1% and 84.6% for imipenem and meropenem, respectively. Lower respiratory tract isolates and isolates from the intensive care unit setting were the least susceptible overall. Resistance rates were typically highest in lower respiratory tract isolates, with the exception of urinary tract isolates, which displayed the highest resistance for levofloxacin. Overall, multidrug-resistant isolates comprised 14.8% of the total sample population.

A Novel Indole Compound That Inhibits Pseudomonas aeruginosa Growth by Targeting MreB Is a Substrate for MexAB-OprM

Drug efflux systems contribute to the intrinsic resistance of Pseudomonas aeruginosa to many antibiotics and biocides and hamper research focused on the discovery and development of new antimicrobial agents targeted against this important opportunistic pathogen. Using a P. aeruginosa PAO1 derivative bearing deletions of opmH, encoding an outer membrane channel for efflux substrates, and four efflux pumps belonging to the resistance nodulation/cell division class including mexAB-oprM, we identified a small-molecule indole-class compound (CBR-4830) that is inhibitory to growth of this efflux-compromised strain. Genetic studies established MexAB-OprM as the principal pump for CBR-4830 and revealed MreB, a prokaryotic actin homolog, as the proximal cellular target of CBR-4830. Additional studies establish MreB as an essential protein in P. aeruginosa, and efflux-compromised strains treated with CBR-4830 transition to coccoid shape, consistent with MreB inhibition or depletion. Resistance genetics further suggest that CBR-4830 interacts with the putative ATP-binding pocket in MreB and demonstrate significant cross-resistance with A22, a structurally unrelated compound that has been shown to promote rapid dispersion of MreB filaments in vivo. Interestingly, however, ATP-dependent polymerization of purified recombinant P. aeruginosa MreB is blocked in vitro in a dose-dependent manner by CBR-4830 but not by A22. Neither compound exhibits significant inhibitory activity against mutant forms of MreB protein that bear mutations identified in CBR-4830-resistant strains. Finally, employing the strains and reagents prepared and characterized during the course of these studies, we have begun to investigate the ability of analogues of CBR-4830 to inhibit the growth of both efflux-proficient and efflux-compromised P. aeruginosa through specific inhibition of MreB function.

Antistaphylococcal Activities of the New Fluoroquinolone JNJ-Q2

ABSTRACT The new broad-spectrum fluoroquinolone JNJ-Q2 displays in vitro activity against Gram-negative and Gram-positive organisms, including methicillin-resistant Staphylococcus aureus (MRSA) and ciprofloxacin-resistant MRSA isolates. Tested with isogenic methicillin-susceptible S. aureus (MSSA) and MRSA strains bearing quinolone-resistant target mutations, JNJ-Q2 displayed MICs ≤ 0.12 μg/ml, values 16- to 32-fold lower than those determined for moxifloxacin. Overexpression of the NorA efflux pump did not impact JNJ-Q2 MICs. Inhibition of S. aureus DNA gyrase and DNA topoisomerase IV enzymes demonstrated that JNJ-Q2 was more potent than comparators against wild-type enzymes and enzymes carrying quinolone-resistant amino acid substitutions, and JNJ-Q2 displayed equipotent activity against both enzymes. In serial-passage studies comparing resistance selection in parallel MRSA cultures by ciprofloxacin and JNJ-Q2, ciprofloxacin readily selected for mutants displaying MIC values of 128 to 512 μg/ml, which were observed within 18 to 24 days of passage. In contrast, cultures passaged in the presence of JNJ-Q2 displayed MICs ≤ 1 μg/ml for a minimum of 27 days of serial passage. A mutant displaying a JNJ-Q2 MIC of 4 μg/ml was not observed until after 33 days of passage. Mutant characterization revealed that ciprofloxacin-passaged cultures with MICs of 256 to 512 μg/ml carried only 2 or 3 quinolone resistance-determining region (QRDR) mutations. Cultures passaged with JNJ-Q2 selection for up to 51 days displayed MICs of 1 to 64 μg/ml and carried between 4 and 9 target mutations. Established in vitro biofilms of wild-type or ciprofloxacin-resistant MRSA exposed to JNJ-Q2 displayed greater decreases in bacterial counts (7 days of exposure produced 4.5 to >7 log10 CFU decreases) than biofilms exposed to ciprofloxacin, moxifloxacin, rifampin, or vancomycin.

Activity of ceftobiprole against Streptococcus pneumoniae isolates exhibiting high-level resistance to ceftriaxone

Tracking Resistance in the US Today (TRUST) 2008 surveillance data showed that 6% of Streptococcus pneumoniae were non-susceptible to ceftriaxone [minimum inhibitory concentration (MIC) ≥ 2 μg/mL] and that 8% of the ceftriaxone-non-susceptible isolates exhibited high-level resistance (MIC ≥ 8 μg/mL). Here we describe the activity of ceftobiprole against ceftriaxone-resistant isolates and characterise the genotypic traits associated with resistance. Thirty isolates with ceftriaxone MICs ≥ 8 μg/mL were analysed by sequencing of penicillin-binding protein (PBP) and murM genes. Sequencing of pbp1a, pbp2b and pbp2x showed nine PBP patterns, with the most common (n=17) being: PBP1a T371S (STMK motif), P432T (SRNVP motif); PBP2b T446A (SSNT motif), A619G (KTGTA motif); and PBP2x T338A and M339F (STMK motif), L364F, I371T, R384G, M400T, L546V (LKSGT motif); six isolates had the same pattern without the PBP2b A619G change. For these 23 isolates, MICs were 8 μg/mL for ceftriaxone, 4-8 μg/mL for penicillin and 0.5-2 μg/mL for ceftobiprole. The remaining seven isolates with higher MICs (ceftriaxone 8-32 μg/mL, penicillin 4-32 μg/mL and ceftobiprole 2-4 μg/mL) had fewer PBP active-site motif substitutions. The majority of isolates (17/30) had murM alleles similar to the wild-type, whilst the rest had alleles reflecting a mosaic structure. No murM alleles were associated with higher MICs. Against these 30 isolates, ceftobiprole was 4-16-fold more active than ceftriaxone. Widely described PBP and MurM substitutions probably account for the high ceftriaxone MICs (8 μg/mL) in the majority of isolates. However, seven isolates with ceftriaxone MICs of 8-32 μg/mL had fewer PBP substitutions in active-site motifs, suggesting either that there is another resistance mechanism or that unique PBP mutations may contribute to high-level β-lactam resistance.

Synergistic Activity of Ceftobiprole and Vancomycin in a Rat Model of Infective Endocarditis Caused by Methicillin-Resistant and Glycopeptide-Intermediate Staphylococcus aureus

ABSTRACT The therapeutic activity of ceftobiprole medocaril, the prodrug of ceftobiprole, was compared to that of vancomycin, daptomycin, and the combination of a subtherapeutic dose of ceftobiprole and vancomycin in a rat model of infective endocarditis due to methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300) or glycopeptide-intermediate Staphylococcus aureus (GISA) (NRS4 and HIP 5836) strains. The minimum bactericidal concentrations of ceftobiprole, vancomycin, and daptomycin at bacterial cell densities similar to those encountered in the cardiac vegetation in the rat endocarditis model were 2, >64, and 8 μg/ml, respectively, for MRSA ATCC 43300 and 4, >64, and 8 μg/ml, respectively, for the GISA strain. Ceftobiprole medocaril administered in doses of 100 mg/kg of body weight given intravenously (i.v.) twice a day (BID) every 8 h (q8h) (equivalent to a human therapeutic dose of ceftobiprole [500 mg given three times a day [TID]) was the most effective monotherapy, eradicating nearly 5 log10 CFU/g MRSA or 6 log10 CFU/g GISA organisms from the cardiac vegetation and had the highest incidence of sterile vegetation compared to the other monotherapies in the endocarditis model. In in vitro time-kill studies, synergistic effects were observed with ceftobiprole and vancomycin on MRSA and GISA strains, and in vivo synergy was noted with combinations of subtherapeutic doses of these agents for the same strains. Additionally, sterile vegetations were achieved in 33 and 60%, respectively, of the animals infected with MRSA ATCC 43300 or GISA NRS4 receiving ceftobiprole-vancomycin combination therapy. In summary, ceftobiprole was efficacious both as monotherapy and in combination with vancomycin in treating MRSA and GISA infections in a rat infective endocarditis model and warrants further evaluation.