Neisseria gonorrhoeae Infection in Women Increases With Rising Gonococcal Burdens in Partners: Chlamydia Coinfection in Women Increases Gonococcal Burden.

BACKGROUND: Likelihood of Neisseria gonorrhoeae infection in women exposed to male sex partners with increasing N. gonorrhoeae burdens and enhancement by Chlamydia trachomatis is not defined. METHODS: We identified men with urethritis and their regular female sex partners. Exposure to N. gonorrhoeae burdens in men was compared in N. gonorrhoeae-infected versus -uninfected partners. Association of N. gonorrhoeae infection in women with burdens in male partners was estimated using logistic regression. Association of C. trachomatis coinfection and N. gonorrhoeae burdens in women adjusted for burdens in male partners was estimated by linear regression. RESULTS: In total, 1816 men were enrolled; 202 had ≥2 partners, 91 who confirmed monogamy and were enrolled; 77% were married. Seventy were partners of N. gonorrhoeae-infected men; 58 (83%) were N. gonorrhoeae infected, 26 (45%) C. trachomatis coinfected. Infected women had partners with 9.3-fold higher N. gonorrhoeae burdens than partners of uninfected women (P = .0041). Association of N. gonorrhoeae infection in women with upper quartiles of N. gonorrhoeae burdens in partners increased (odds ratios ≥ 2.97)compared to the first quartile (P = .032). N. gonorrhoeae burdens in C. trachomatis-coinfected women were 2.82-fold higher than in C. trachomatis-uninfected women (P = .036). CONCLUSIONS: N. gonorrhoeae infections increased in women whose partners were infected with higher N. gonorrhoeae burdens. C. trachomatis coinfection was associated with increased N. gonorrhoeae burdens in women.

Frequency and Mechanism of Spontaneous Resistance to Sulbactam Combined with the Novel ß-Lactamase Inhibitor ETX2514 in Clinical Isolates of Acinetobacter baumannii.

The novel diazabicyclooctenone ETX2514 is a potent, broad-spectrum serine ß-lactamase inhibitor that restores sulbactam activity against resistant Acinetobacter baumannii The frequency of spontaneous resistance to sulbactam-ETX2514 in clinical isolates was found to be 7.6 × 10-10 to <9.0 × 10-10 at 4× MIC and mapped to residues near the active site of penicillin binding protein 3 (PBP3). Purified mutant PBP3 proteins demonstrated reduced affinity for sulbactam. In a sulbactam-sensitive isolate, resistance also mapped to stringent response genes associated with resistance to PBP2 inhibitors, suggesting that in addition to ß-lactamase inhibition, ETX2514 may enhance sulbactam activity in A. baumannii via inhibition of PBP2.

Elucidation of Mechanisms of Ceftazidime Resistance among Clinical Isolates of Pseudomonas aeruginosa by Using Genomic Data.

Ceftazidime is one of the few cephalosporins with activity against Pseudomonas aeruginosa Using whole-genome comparative analysis, we set out to determine the prevalent mechanism(s) of resistance to ceftazidime (CAZ) using a set of 181 clinical isolates. These isolates represented various multilocus sequence types that consisted of both ceftazidime-susceptible and -resistant populations. A presumptive resistance mechanism against ceftazidime was identified in 88% of the nonsusceptible isolates using this approach.

Homologous Recombination within Large Chromosomal Regions Facilitates Acquisition of ß-Lactam and Vancomycin Resistance in Enterococcus faecium.

The transfer of DNA between Enterococcus faecium strains has been characterized both by the movement of well-defined genetic elements and by the large-scale transfer of genomic DNA fragments. In this work, we report on the whole-genome analysis of transconjugants resulting from mating events between the vancomycin-resistant E. faecium C68 strain and the vancomycin-susceptible D344RRF strain to discern the mechanism by which the transferred regions enter the recipient chromosome. Vancomycin-resistant transconjugants from five independent matings were analyzed by whole-genome sequencing. In all cases but one, the penicillin binding protein 5 (pbp5) gene and the Tn5382 vancomycin resistance transposon were transferred together and replaced the corresponding pbp5 region of D344RRF. In one instance, Tn5382 inserted independently downstream of the D344RRF pbp5 gene. Single nucleotide variant (SNV) analysis suggested that entry of donor DNA into the recipient chromosome occurred by recombination across regions of homology between donor and recipient chromosomes, rather than through insertion sequence-mediated transposition. The transfer of genomic DNA was also associated with the transfer of C68 plasmid pLRM23 and another putative plasmid. Our data are consistent with the initiation of transfer by cointegration of a transferable plasmid with the donor chromosome, with subsequent circularization of the plasmid-chromosome cointegrant in the donor prior to transfer. Entry into the recipient chromosome most commonly occurred across regions of homology between donor and recipient chromosomes.

Multiyear, Multinational Survey of the Incidence and Global Distribution of Metallo-ß-Lactamase-Producing Enterobacteriaceae and Pseudomonas aeruginosa.

Metallo-ß-lactamases (MBLs) hydrolyze all classes of ß-lactams except monobactams and are not inhibited by classic serine ß-lactamase inhibitors. Gram-negative pathogens isolated from patient infections were collected from 202 medical centers in 40 countries as part of a global surveillance study from 2012 to 2014. Carbapenem-nonsusceptible Enterobacteriaceae and Pseudomonas aeruginosa were characterized for bla genes encoding VIM, IMP, NDM, SPM, and GIM variants using PCR and sequencing. A total of 471 MBL-positive isolates included the following species (numbers of isolates are in parentheses): P. aeruginosa (308), Klebsiella spp. (85), Enterobacter spp. (39), Proteeae (16), Citrobacter freundii (12), Escherichia coli (6), and Serratia marcescens (5) and were submitted by sites from 34 countries. Of these, 69.6% were collected in 9 countries (numbers of isolates are in parentheses): Russia (72), Greece (61), Philippines (54), Venezuela (29), and Kuwait, Nigeria, Romania, South Africa, and Thailand (20 to 25 isolates each). Thirty-two different MBL variants were detected (14 VIM, 14 IMP, and 4 NDM enzymes). Seven novel MBL variants were encountered in the study, each differing from a previously reported variant by one amino acid substitution: VIM-42 (VIM-1 [V223I]), VIM-43 (VIM-4 [A24V]), VIM-44 (VIM-2 [K257N]), VIM-45 (VIM-2 [T35I]), IMP-48 (IMP-14 [I69T]), IMP-49 (IMP-18 [V49F]), and NDM-16 (NDM-1 [R264H]). The in vitro activities of all tested antibiotics against MBL-positive Enterobacteriaceae were significantly reduced with the exception of that of aztreonam-avibactam (MIC90, 0.5 to 1 µg/ml), whereas colistin was the most effective agent against MBL-positive P. aeruginosa isolates (>97% susceptible). Although the global percentage of isolates encoding MBLs remains relatively low, their detection in 12 species, 34 countries, and all regions participating in this surveillance study is concerning.

Small-molecule inhibitors of gram-negative lipoprotein trafficking discovered by phenotypic screening.

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.

The resistome of Pseudomonas aeruginosa in relationship to phenotypic susceptibility.

Many clinical isolates of Pseudomonas aeruginosa cause infections that are difficult to eradicate due to their resistance to a wide variety of antibiotics. Key genetic determinants of resistance were identified through genome sequences of 390 clinical isolates of P. aeruginosa, obtained from diverse geographic locations collected between 2003 and 2012 and were related to microbiological susceptibility data for meropenem, levofloxacin, and amikacin. ß-Lactamases and integron cassette arrangements were enriched in the established multidrug-resistant lineages of sequence types ST111 (predominantly O12) and ST235 (O11). This study demonstrates the utility of next-generation sequencing (NGS) in defining relevant resistance elements and highlights the diversity of resistance determinants within P. aeruginosa. This information is valuable in furthering the design of diagnostics and therapeutics for the treatment of P. aeruginosa infections.

Molecular mechanisms of sulbactam antibacterial activity and resistance determinants in Acinetobacter baumannii.

Sulbactam is a class A ß-lactamase inhibitor with intrinsic whole-cell activity against certain bacterial species, including Acinetobacter baumannii. The clinical use of sulbactam for A. baumannii infections is of interest due to increasing multidrug resistance in this pathogen. However, the molecular drivers of its antibacterial activity and resistance determinants have yet to be precisely defined. Here we show that the antibacterial activities of sulbactam vary widely across contemporary A. baumannii clinical isolates and are mediated through inhibition of the penicillin-binding proteins (PBPs) PBP1 and PBP3, with very low frequency of resistance; the rare pbp3 mutants with high levels of resistance to sulbactam are attenuated in fitness. These results support further investigation of the potential clinical utility of sulbactam.

Characterization of the novel DNA gyrase inhibitor AZD0914: low resistance potential and lack of cross-resistance in Neisseria gonorrhoeae.

The unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae infections is significant and increasing, as rapidly emerging resistance in this pathogen is threatening to eliminate the currently available treatment options. AZD0914 is a novel bacterial gyrase inhibitor that possesses potent in vitro activities against isolates with high-level resistance to ciprofloxacin and extended-spectrum cephalosporins, and it is currently in clinical development for the treatment of N. gonorrhoeae infections. The propensity to develop resistance against AZD0914 was examined in N. gonorrhoeae and found to be extremely low, a finding supported by similar studies with Staphylococcus aureus. The genetic characterization of both first-step and second-step mutants that exhibited decreased susceptibilities to AZD0914 identified substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary target of AZD0914 and providing differentiation from fluoroquinolones. The analysis of available bacterial gyrase and topoisomerase IV structures, including those bound to fluoroquinolone and nonfluoroquinolone inhibitors, has allowed the rationalization of the lack of cross-resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data also indicate that the topoisomerase inhibition mechanisms are subtly different between N. gonorrhoeae and other bacterial species. Taken together, these data support the progression of AZD0914 as a novel treatment option for the oral treatment of N. gonorrhoeae infections.

Pharmacodynamic Profiling of a Siderophore-Conjugated Monocarbam in Pseudomonas aeruginosa: Assessing the Risk for Resistance and Attenuated Efficacy.

The objective of this study was to investigate the risk of attenuated efficacy due to adaptive resistance for the siderophore-conjugated monocarbam SMC-3176 in Pseudomonas aeruginosa by using a pharmacokinetic/pharmacodynamic (PK/PD) approach. MICs were determined in cation-adjusted Mueller-Hinton broth (MHB) and in Chelex-treated, dialyzed MHB (CDMHB). Spontaneous resistance was assessed at 2× to 16× the MIC and the resulting mutants sequenced. Efficacy was evaluated in a neutropenic mouse thigh model at 3.13 to 400 mg/kg of body weight every 3 h for 24 h and analyzed for association with free time above the MIC (fT>MIC). To closer emulate the conditions of the in vivo model, we developed a novel assay testing activity mouse whole blood (WB). All mutations were found in genes related to iron uptake: piuA, piuC, pirR, fecI, and pvdS. Against four P. aeruginosa isolates, SMC-3176 displayed predictable efficacy corresponding to the fT>MIC using the MIC in CDMHB (R(2) = 0.968 to 0.985), with stasis to 2-log kill achieved at 59.4 to 81.1%. Efficacy did not translate for P. aeruginosa isolate JJ 4-36, as the in vivo responses were inconsistent with fT>MIC exposures and implied a threshold concentration that was greater than the MIC. The results of the mouse WB assay indicated that efficacy was not predictable using the MIC for JJ 4-36 and four additional isolates, against which in vivo failures of another siderophore-conjugated ß-lactam were previously reported. SMC-3176 carries a risk of attenuated efficacy in P. aeruginosa due to rapid adaptive resistance preventing entry via the siderophore-mediated iron uptake systems. Substantial in vivo testing is warranted for compounds using the siderophore approach to thoroughly screen for this in vitro-in vivo disconnect in P. aeruginosa.

Molecular characterization of MRSA isolates bracketing the current EUCAST ceftaroline-susceptible breakpoint for Staphylococcus aureus: the role of PBP2a in the activity of ceftaroline.

OBJECTIVES: The objectives of this study were to characterize contemporary MRSA isolates and understand the prevalence and impact of sequence variability in PBP2a on ceftaroline susceptibility. METHODS: A total of 184 MRSA isolates collected from 28 countries were collected and characterized. RESULTS: WT PBP2a proteins were found in MRSA distributed evenly over the ceftaroline MIC range of 0.5-2 mg/L (n=56). PBP2a variations found in 124 isolates fell into two categories: (i) 12 isolates contained a substitution in the transpeptidase pocket located in the penicillin-binding domain and exhibited significantly decreased ceftaroline susceptibility (typically 8 mg/L); and (ii) isolates with substitutions in the non-penicillin-binding domain (nPBD) in a region proposed to be functionally important for cell wall biogenesis. The majority (71%) of isolates containing only nPBD variations were inhibited by 2 mg/L ceftaroline, 23% by ≤1 mg/L and 6% by 4 mg/L. These data suggest that the WT MRSA distribution extends beyond the current EUCAST and CLSI susceptible breakpoints and includes isolates inhibited by 2 mg/L ceftaroline. SCCmec type IV was the predominant type in the ceftaroline-susceptible population (68%), whereas it only represented 6% of the non-susceptible population. The variations of MLST lineages were fewer among the non-susceptible group. CONCLUSIONS: This study suggests that MRSA populations with a WT PBP2a and those with nPBD variations overlap significantly and that PBP2a sequence-independent factors contribute to ceftaroline susceptibility. Whereas characterization of isolates with a ceftaroline MIC of 2 mg/L enriched for isolates with nPBD variations, it was not a discrete population. In contrast, the rare isolates containing a substitution in the transpeptidase-binding pocket were readily differentiated.

Pharmacokinetics/pharmacodynamics of a ß-lactam and ß-lactamase inhibitor combination: a novel approach for aztreonam/avibactam.

OBJECTIVES: The combination of aztreonam/avibactam has promising activity against MDR Gram-negative pathogens producing metallo-ß-lactamases (MBLs), such as New Delhi MBL-1. Pharmacokinetic (PK)/pharmacodynamic (PD) understanding of this combination is critical for optimal clinical dose selection. This study focuses on the determination of an integrated PK/PD approach for aztreonam/avibactam across multiple clinical Enterobacteriaceae strains. METHODS: Six clinical Enterobacteriaceae isolates expressing MBLs and ESBLs were studied in an in vitro hollow-fibre infection model (HFIM) using various dosing regimens simulating human-like PK for aztreonam/avibactam. The neutropenic murine thigh infection model was used for in vivo validation against two bacterial strains. RESULTS: MIC values of aztreonam/avibactam for the isolates ranged from 0.125 to 8 mg/L. Using a constant infusion of avibactam at 4 mg/L, the aztreonam PK/PD index was observed as % fT >MIC. Studies performed in the presence of a fixed dose of aztreonam revealed that the efficacy of avibactam correlates best with percentage of time above a critical threshold concentration of 2-2.5 mg/L. These conclusions translated well to the efficacy observed in the murine thigh model, demonstrating in vivo validation of the in vitro PK/PD target. CONCLUSIONS: PK/PD evaluations for aztreonam/avibactam in HFIM yielded a single target across strains with a wide MIC range. This integrated approach could be easily applied for forecasting clinically efficacious doses for ß-lactam/ß-lactamase inhibitor combinations.

Selection and molecular characterization of ceftazidime/avibactam-resistant mutants in Pseudomonas aeruginosa strains containing derepressed AmpC.

OBJECTIVES: Pseudomonas aeruginosa is an important nosocomial pathogen that can cause a wide range of infections resulting in significant morbidity and mortality. Avibactam, a novel non-ß-lactam ß-lactamase inhibitor, is being developed in combination with ceftazidime and has the potential to be a valuable addition to the treatment options for the infectious diseases practitioner. We compared the frequency of resistance development to ceftazidime/avibactam in three P. aeruginosa strains that carried derepressed ampC alleles. METHODS: The strains were incubated in the presence of increasing concentrations of ceftazidime with a fixed concentration (4 mg/L) of avibactam to calculate the frequency of spontaneous resistance. The mutants were characterized by WGS to identify the underlying mechanism of resistance. A representative mutant protein was characterized biochemically. RESULTS: The resistance frequency was very low in all strains. The resistant variants isolated exhibited ceftazidime/avibactam MIC values that ranged from 64 to 256 mg/L. All of the mutants exhibited changes in the chromosomal ampC gene, the majority of which were deletions of various sizes in the Ω-loop region of AmpC. The mutant enzyme that carried the smallest Ω-loop deletion, which formed a part of the avibactam-binding pocket, was characterized biochemically and found to be less effectively inhibited by avibactam as well as exhibiting increased hydrolysis of ceftazidime. CONCLUSIONS: The development of high-level resistance to ceftazidime/avibactam appears to occur at low frequency, but structural modifications in AmpC can occur that impact the ability of avibactam to inhibit the enzyme and thereby protect ceftazidime from hydrolysis.

Characterization of a respiratory syncytial virus L protein inhibitor.

The respiratory syncytial virus (RSV) L protein is a viral RNA-dependent RNA polymerase that contains multiple enzyme activities required for RSV replication. The RSV L inhibitors described in literature are limited by their cytotoxicity or the lack of RSV B subtype coverage. Here, we characterize a new RSV L inhibitor with strong antiviral activity against both RSV A and B subtypes and no detectable cytotoxicity. This compound, AZ-27, was equally active against RSV live viruses and subgenomic replicons and demonstrated advantages over other classes of RSV inhibitors in time-of-addition and cell line dependency studies. Resistance studies identified a dominant mutation in the putative capping enzyme domain of L protein, which conferred strong resistance to the AZ-27 series but not other classes of RSV inhibitors, supporting RSV L protein as the direct target for AZ-27. This novel and broad-spectrum RSV L polymerase inhibitor may pave the way toward an efficacious RSV therapeutic and provide a new tool for interrogation of the L protein function.

A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria.

Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.

Analysis of Staphylococcus aureus clinical isolates with reduced susceptibility to ceftaroline: an epidemiological and structural perspective.

OBJECTIVES: Ceftaroline, approved in Europe in 2012, has activity against methicillin-resistant Staphylococcus aureus (MRSA), with MIC90 values of 1-2 mg/L depending on geographical location. During a global 2010 surveillance programme, conducted prior to the European launch, 4 S. aureus isolates, out of 8037 tested, possessing ceftaroline MIC values of >2 mg/L were identified. The objective of this study was to characterize these four isolates to elucidate the mechanism of ceftaroline resistance. METHODS: MIC determinations were performed using broth microdilution and whole genome sequencing was performed to enable sequence-based analyses. RESULTS: The only changes in proteins known to be required for full expression of methicillin resistance that correlated with the ceftaroline MIC were in penicillin-binding protein 2a (PBP2a). Isolates with a ceftaroline MIC of 2 mg/L had a Glu239Lys mutation in the non-penicillin-binding domain whereas the four isolates with ceftaroline MIC values of 8 mg/L carried an additional Glu447Lys mutation in the penicillin-binding domain. The impact of these mutations was analysed using the known X-ray structure of S. aureus PBP2a and a model for ceftaroline resistance proposed. Analysis of the core genomes showed that the isolates with reduced susceptibility to ceftaroline were epidemiologically related. CONCLUSIONS: Mutations in PBP2a can affect the activity of ceftaroline against MRSA. Although a rare event, based on surveillance studies, it appears a first-step change in the non-penicillin-binding domain together with a second-step in the penicillin-binding domain may result in elevation of the ceftaroline MIC to >2 mg/L.

Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria.

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-ß-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four ß-lactamase genes (bla KPC, bla NDM-1, bla IMP-1 group, and bla VIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six ß-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four ß-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four ß-lactamases.

Development of a Novel Loop-Mediated Isothermal Amplification Method to Detect Guiana Extended-Spectrum (GES) ß-Lactamase Genes in Pseudomonas aeruginosa.

Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalized patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) ß-lactamase-producing strains are of growing concern. Several genotypes of the GES ß-lactamase gene (bla GES) include a single missense mutation, a change from G to A at nucleotide position 493 (G493A) that changes glycine to serine; the mutant enzyme exhibits carbapenemase activity. Rapid and reliable identification of drug-resistance is important in clinical settings; however, culture methods remain the gold standard. Conventional and real-time PCR cannot identify carbapenemase-producing genotypes, and direct DNA sequencing is essential. We established a novel loop-mediated isothermal amplification (LAMP) method to detect various genotypes of bla GES and another LAMP method to discriminate carbapenemase genotypes of bla GES. We evaluated the two assays using clinical P. aeruginosa strains. Two primer sets targeting bla GES (GES-LAMP) and the point mutation (Carba-GES-LAMP) were designed and evaluated for specificity and sensitivity. The detection limit of the GES-LAMP method was assessed using purified DNA and DNA-spiked clinical samples (urine, sputum, and blood). To determine the clinical usefulness of the methods, we used different (genotypically and phenotypically) P. aeruginosa clinical isolates, collected from diverse geographical locations between 2003 and 2012. The novel LAMP assay targeting bla GES was highly specific. The detection limit was 10 DNA copies per reaction; the assay was 10-fold more sensitive than conventional PCR. The LAMP assay detected bla GES with high sensitivity in all DNA-spiked samples; PCR did not detect bla GES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing bla GES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two bla GES positive organisms with carbapenemase activity. To the best of our knowledge, this is the first report of the GES ß-lactamase gene detection assay using the LAMP method. Our new assays effectively detect bla GES and critical unique mutations.

Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis.

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Arthroscopic treatment of the arthritic elbow due to primary radiocapitellar arthritis.

PURPOSE: To retrospectively compare patients who received radial head excision for early, isolated radiocapitellar arthritis with those who had delayed surgery for the same problem in order to analyze risk factors for progression of the arthritis. Isolation of risk factors for progression should allow guidelines for early excision and perhaps prevent progression to panarticular arthritis. TYPE OF STUDY: Retrospective analysis of a group of patients with radiocapitellar arthritis. METHODS: From 1995 to 2001, 36 consecutive patients with arthritic damage to the radiocapitellar joint were treated with arthroscopic debridement and radial head excision. Twenty-eight of the 36 underwent concurrent arthroscopic modification of the Outerbridge-Kashiwagi procedure because of the additional presence of ulnohumeral arthritis. All patients were re-examined 18 to 91 months (mean, 52 months) after the procedure and evaluated using the Andrews-Carson (A-C) elbow rating system. RESULTS: In patients who underwent radial head excision alone, flexion increased 29 degrees, extension 38 degrees, with an increase in total arc of motion of 62 degrees. In patients who underwent radial head excision and ulnohumeral arthroplasty, postoperative flexion increased 19 degrees, extension 27 degrees, with an increase in total arc of motion of 46 degrees. The difference between the 2 groups of patients was significant (P = .002). The average preoperative A-C score for patients undergoing radial head excision alone was 72 and the average postoperative score was 170. The average preoperative score for patients undergoing radial head excision and ulnohumeral arthroplasty was 92 and the average postoperative score was 150. Two patients who underwent the combined procedure had to return to surgery: 1 for a contracture release and 1 for radial head replacement secondary to intractable pain. They both did well subsequently. CONCLUSIONS: Patients undergoing radial head excision alone increased their average A-C score almost 100 points. Those undergoing both procedures increased their score an average of 58 points. In addition, those undergoing only radial head excision had a 20-point higher average overall postoperative A-C rating than those undergoing both procedures. Patients who underwent radial head excision alone had a greater return of range of motion and fewer postoperative complications than those who underwent the combined procedure. LEVEL OF EVIDENCE: Level IV.