Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-β-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four β-lactamase genes (blaKPC, blaNDM-1, blaIMP-1 group, and blaVIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six β-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 β-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 β-lactamases.

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.

FMX101 4% minocycline foam (FMX101 4%) is a novel, topical minocycline formulation for treatment of acne vulgaris. We report that FMX101 4% had an MIC90 of 0.25 μg/ml and was ≥4-fold more active than comparator antimicrobials against a panel of 98 clinical Cutibacterium acnes isolates. The panel was diverse by clonal complex and sequence type, having 20 novel multi-locus sequence types including clonal complexes and sequence types associated with acne (CC1, CC3, and CC4; ST1 and ST3). Some isolates were phenotypically resistant to clindamycin (6.1%), erythromycin (14.3%), and tetracycline (2.0% intermediate resistance). Six isolates (6.4%) carried a mutation in the quinolone resistance-determining region of gyrA. With C. acnes, spontaneous resistance to FMX101 4% occurred at frequencies ranging from ≤5 × 10-9 to <1 × 10-8; mutations were identified in rpsJ, a gene encoding 30S ribosomal protein S10. No mutant exhibited a minocycline MIC above 0.5 μg/ml. No second-step mutation in previously isolated mutants or strains containing rpsJ ± 16S rRNA mutations was detected following minocycline challenge. Minocycline retained antibacterial activity against C. acnes over 15 multiple passages; thus, no selective growth advantage for minocycline-resistant mutants occurred under the experimental conditions. FMX101 4% has the potential to retain the favorable resistance profile of minocycline in diverse C. acnes isolates while providing the benefits of a topical formulation for treatment of acne vulgaris.

Abstract This chapter explores the purposes of the doctrine on recognition of belligerency. Whilst these purposes range from the evidently humanitarian to the explicitly political, they were nevertheless primarily expressed via the language and application of law. The means by which humanity was to be encouraged, for example, was to invoke de jure application of the Law of War as to means and methods, treatment of prisoners of war, and conduct vis-à-vis neutral third states. The means by which political ends were served included the application of the law of neutrality to characterize maritime interactions between third states on the one hand, and the conflict state and belligerent non-state armed groups (NSAGs) on the other, and employing recognition of belligerency as a middle path between assuaging public agitation in support of insurgents and the counter-veiling diplomatic imperative to maintain the veneer of non-intervention against fellow states. However, the doctrine also evinced a level of legal novelty and progressive development in response to perceived needs, such as the limited status and attributes afforded a recognized belligerent NSAG in terms of raising funds, taxing activity, and taking responsibility for treatment of and consequences for third state nationals.

Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalized patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) β-lactamase-producing strains are of growing concern. Several genotypes of the GES β-lactamase gene (blaGES) 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 blaGES and another LAMP method to discriminate carbapenemase genotypes of blaGES. We evaluated the two assays using clinical P. aeruginosa strains. Two primer sets targeting blaGES (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 blaGES 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 blaGES with high sensitivity in all DNA-spiked samples; PCR did not detect blaGES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing blaGES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two blaGES positive organisms with carbapenemase activity. To the best of our knowledge, this is the first report of the GES β-lactamase gene detection assay using the LAMP method. Our new assays effectively detect blaGES and critical unique mutations.

In April 1912, the third Irish Home Rule Bill was introduced in the British House of Commons. The north of Ireland erupted with protests opposing Home Rule for Ireland, claiming it would mean “Rome Rule.” Ulster Protestants were particularly opposed to Irish Home Rule and made certain that its implementation would fail. Orange Canadians were interested in events in Ireland to such an extent many became active participants in those events, through moral, material, and even physical support of the Ulster unionist opposition to the implementation of Irish Home Rule. This Orange Canadian response and demonstration of fidelity with their Irish co-religionists is often overlooked by historians of the Canadian Order. Canadian Orangemen maintained strong connections with their brethren in Ireland, and viewed themselves as a North-American counterweight to the strong support Irish nationalists found in the United States. These determined expressions of support for the stance taken by their Orange brethren in Ireland continued over such a long period of time, and were demonstrated with such intensity, that these expressions of fidelity and support to Irish Protestants generally should not be viewed as a fleeting chant, or as anachronistic, but should be viewed in the larger context of transnational Orange solidarity and brotherhood.

Background:Bispecific antibody MEDI3902, targeting the Pseudomonas aeruginosa type 3 secretion system (PcrV) and Psl exopolysaccharide, is currently in phase 2b development for prevention of nosocomial pneumonia in patients undergoing mechanical ventilation. We surveyed a diverse collection of isolates to study MEDI3902 epitope conservation and protective activity.Methods:P. aeruginosa clinical isolates (n = 913) were collected from diverse patients and geographic locations during 2003-2014. We conducted whole-genome sequencing; performed PcrV and Psl expression analyses via immunoblotting and enzyme-linked immunosorbent assay, respectively; performed crystallography to determine the MEDI3902 PcrV epitope, using anti-PcrV Fab and PcrV components (resolved at 2.8 Å); and evaluated MEDI3902 protective activity against select isolates in vitro and in vivo.Results:Intact psl operon and pcrV genes were present in 94% and 99% of isolates, respectively, and 99.9% of isolates contained at least one of the genetic elements. Anti-Psl binding was confirmed in tested isolates harboring a complete Psl operon or lacking nonessential psl genes. We identified 46 PcrV variant sequences, and MEDI3902-PcrV contact residues were preserved. MEDI3902 maintained potent in vivo activity against various strains, including strains expressing only a single target.Conclusions:Psl and PcrV are highly prevalent in global clinical isolates, suggesting MEDI3902 can mediate broad coverage against P. aeruginosa.

The May 1961 'Red Crusader' incident, involving a British trawler and a Danish warship, has - along with the I'm Alone incident and the 'MV Saiga' case - become a milestone in the development and c...

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.

ABSTRACT The novel diazabicyclooctenone ETX2514 is a potent, broad-spectrum serine β-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 β-lactamase inhibition, ETX2514 may enhance sulbactam activity in A. baumannii via inhibition of PBP2.

CAZ-AVI has been evaluated in Phase 3 trials for the treatment of complicated urinary tract infections, complicated intra-abdominal infections, and hospital-acquired pneumonia, including ventilator-associated pneumonia. This study presents the β-lactamase characterization of baseline PSA recovered from patients enrolled in five Phase 3 trials of CAZ-AVI. 189 baseline PSA isolates (one per patient) were included (19 countries). Isolates met the β-lactamase MIC screening criteria, and displayed CAZ MIC ≥16 µg/ml and/or carbapenem MIC ≥8 µg/ml. Susceptibility (S) testing was centrally performed by CLSI methods. Isolates underwent microarray-based assay, PCR/sequencing for screening of ESBL and carbapenemases (CARB), and qRT-PCR for determination of chromosomal AmpC expression. Isolates showed low S rates for CAZ (30.7%S; MIC50/90, 32/>64 µg/ml), meropenem (30.2%S; MIC50/90, 8/>8 µg/ml) and piperacillin-tazobactam (28.8%S; MIC50/90, 64/>128 µg/ml). In all, 27.5% (52/189) of isolates were CAZ-AVI resistant (R) and 36.5% (19/52) of these carried metallo-β-lactamases; 12 isolates carried IMP alleles, which were detected in China (blaIMP-25), Czech Republic (blaIMP-7), Mexico (blaIMP-18 and -56), Ukraine (blaIMP-1), and Vietnam (blaIMP-26), while 2 blaVIM-1-carrying PSA were found in Ukraine and 5 blaVIM-2 in Romania and Russia. Among CAZ-nonS PSA (69.3%; 131/189), 45.8% (60/131) showed overexpression of AmpC with or without blaOXA-like (OXA-2, -10 or -17), PSE-1, PER-1, IMP-56 or VIM-like (VIM-1 or -2) enzymes, 31.3% (41/131) had numerous combinations of CARB and ESBL enzymes, and 22.9% (30/131) had no β-lactamase genes detected. In total, 58/189 (30.7%) CAZ-S PSA were selected due to high MIC results for carbapenems. Among these 58 isolates, only five carried ESBL genes (blaOXA-2, blaPSE-1 and blaOXA-74). A great proportion (45.8%) of CAZ-nonS PSA was hyper-producers of the chromosomal AmpC enzyme. CAZ-nonS isolates also carried various combinations of CARB and ESBL genes, while CAZ-S and carbapenem-R PSA likely had non-enzymatic β-lactam resistance mechanisms. Funding: This study was sponsored by AstraZeneca (AZ). The AZ product ceftazidime-avibactam was acquired by Pfizer in December 2016. R. E. Mendes, AstraZeneca: Research Contractor, Research grant. M. Castanheira, AstraZeneca: Research Contractor, Research grant. L. N. Woosley, AstraZeneca: Research Contractor, Research grant. G. G. Stone, AstraZeneca: Formerly an employee and shareholder at time of study, salary and shareholder. Pfizer Inc.: Employee, Salary. R. Mclaughlin, AstraZeneca: Employee, Salary. P. Bradford, AstraZeneca: Formerly an employee and shareholder at time of study, salary and shareholder. R. K. Flamm, AstraZeneca: Research Contractor, Research grant

Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of β-lactamases, enzymes that inactivate β-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new β-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine β-lactamase inhibitors that potently inhibit clinically relevant class A, C and D β-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of β-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam–ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need. Development of a broad-spectrum β-lactamase inhibitor capable of combatting multidrug-resistant pathogens, including Acinetobacter baumannii.

ISSUE:This qualitative study gathered the opinions of healthcare employers to better understand the importance, benefits, obstacles, and evolving issues related to allied health (AH) clinical education from the employers' perspective, with the goal to identify opportunities to strengthen and improve clinical-educational partnerships.METHOD:Member deans of the Association of Schools of Allied Health Professions (ASAHP) provided names and contact information of employers that routinely educate their students. Interviews were scheduled with employers who responded to Clinical Education Task Force (CETF) invitation. Twenty-one interviews were conducted by CETF members in early 2016 and analyzed utilizing qualitative software.OUTCOMES:Themes included benefits of working with students and hiring trainees, and obstacles of time and effort required to host students. A trend was noted in gaps between educational preparation and clinical performance. Recent changes highlighted increased technology and regulation, while anticipated changes included more focus on learning on site, longer clinical experiences, and payment for clinical education.CONCLUSION:Collaboration between educators and employers is essential to ensure that curriculum and outcomes match the needs of the field and effectively prepare students as entry-level clinicians.

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.

ABSTRACT 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 Tn 5382 vancomycin resistance transposon were transferred together and replaced the corresponding pbp5 region of D344RRF. In one instance, Tn 5382 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.

181. Loop-Mediated Isothermal Amplification Assay for β-Lactamase Identification on Clinical Isolates of Pseudomonas aeruginosa Mitsuko Seki, DDS, PhD; Daisuke Omagari, DDS, PhD; Paul Kilgore, MD, MPH; Dong Wook Kim, PhD; Tatsushi Adachi, MSc; Robert Mclaughlin, PhD; Humphrey Gardner, MD, FCAP; Abdulbaset Salim, MD, MPH; Satoshi Hayakawa, MD, PhD; Nihon University, Tokyo, Japan; Wayne State University, Detroit, MI; Hanyang University, Ansan, South Korea; Kaneka Co. Ltd, Takasago, Japan; AstraZeneca, Waltham, Massachusetts

A large percentage of Pseudomonas aeruginosa clinical isolates have been noted to be resistant to carbapenems due to loss of function of the OprD porin, the primary mechanism of entry for carbapenems. Such modifications also substantially abolish the organism's ability to transport arginine. Here we report the identification of an in-frame deletion in oprD which confers carbapenem resistance but is expressed and retains the ability to transport arginine.

Background.Multidrug-resistant (MDR) Acinetobacter baumannii infections are of great concern due to high mortality rates and limited number of treatment options.β-lactamase (BL) expression, especially class D, is an important resistance mechanism in this organism.The novel BL inhibitor ETX2514 has potent activity against class A, C, and D serine BLs.The MIC 90 of sulbactam (SUL) in the presence of ETX2514 is 4 mg/L against a large, globally diverse set of MDR A. baumannii clinical isolates from 2014.In this study, we investigate the mechanism of synergy of this combination alone or in the presence of imipenem (IPM) or meropenem (MEM), whose spectra of target inhibition vary across bacterial species Methods.MICs were performed according to CLSI guidelines.The frequency of resistance (FOR) to SUL/ETX2514 was determined in several clinical isolates of A. baumannii.Resistant mutants were analyzed by whole-genome sequencing.Morphological changes were examined by microscopy.PBP acylation rates were determined by competition with BOCILLIN FL in fluorescence polarization assays.Results.The MIC 50 and MIC 90 of relevant combinations against 598 contemporary isolates of A. baumannii are shown in the table. Compound(s)MIC 50 MIC 90

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. beta-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.