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A total of 10,890 bacterial isolates of Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus and Escherichia coli isolated as etiological agents from dairy cows with mastitis by 29 veterinary laboratories across North America between 2011 and 2022 were tested for in vitro antimicrobial susceptibility by broth microdilution to ampicillin, cefoperazone, ceftiofur, cephalothin, erythromycin, oxacillin, penicillin-novobiocin and pirlimycin according to CLSI standards. Using available clinical breakpoints, antimicrobial resistance among S. dysgalactiae (n = 2406) was low for penicillin-novobiocin (0% resistance), ceftiofur (0.1%), erythromycin (3.2%) and pirlimycin (4.6%). Among S. uberis (n = 2398), resistance was low for ampicillin (0%) and ceftiofur (0.2%) and moderate for erythromycin (11.9%) and pirlimycin (18.4%). For S. aureus (n = 3194), resistance was low for penicillin-novobiocin (0%), ceftiofur (0.1%), oxacillin (0.2%), erythromycin (0.7%), cefoperazone (1.2%) and pirlimycin (2.8%). For E. coli (n = 2892), resistance was low for ceftiofur (2.8%) and cefoperazone (3.4%) and moderate for ampicillin (9.2%). Overall, the results indicate that mastitis pathogens in the United States and Canada have not shown any substantial changes in the in vitro susceptibility to antimicrobial drugs over the 12 years of the study, or among that of the proceeding survey from 2002-2010. The data support the conclusion that resistance to common antimicrobial drugs among mastitis pathogens, even to drugs that have been used in dairies for mastitis management for many years, continues to remain low., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The emergence of antimicrobial resistance in human and veterinary bacterial pathogens has led to concerns regarding the use of antimicrobials in veterinary medicine. Consequently, regulatory agencies have developed procedures for assessing the risk associated with the use of a specific antimicrobial as part of the drug approval process. Due consideration for the importance (priority categorization) of the antimicrobial to human medicine is part of this risk assessment process. Additionally, nongovernmental organizations have developed antimicrobial categorization schemes to protect the use and effectiveness of these medicines. However, the goals and methods of the various categorization schemes vary, resulting in final categorizations that are different. Although harmonizing these schemes would bring clarity to antimicrobial resistance discussions and policy, it has the disadvantage of not accounting for regional antimicrobial resistance and use, potentially removing effective medicines from clinical use in situations where they are wholly appropriate. Antimicrobials should be classified in a One Health manner, where both physician and veterinarian share the responsibility for antimicrobial use. The purpose of this article is to summarize current antimicrobial categorization schemes using illustrative examples to highlight differences and provide perspectives on the impact of the current schemes and future directions., (© 2020 Zoetis LLC. Journal of Veterinary Pharmacology and Therapeutics published by John Wiley & Sons Ltd.)

Background: Mannheimia haemolytica strains isolated from North American cattle have been classified into two genotypes (1 and 2). Although members of both genotypes have been isolated from the upper and lower respiratory tracts of cattle with or without bovine respiratory disease (BRD), genotype 2 strains are much more frequently isolated from diseased lungs than genotype 1 strains. The mechanisms behind the increased association of genotype 2 M. haemolytica with BRD are not fully understood. To address that, and to search for interventions against genotype 2 M. haemolytica, complete, closed chromosome assemblies for 35 genotype 1 and 34 genotype 2 strains were generated and compared. Searches were conducted for the pan genome, core genes shared between the genotypes, and for genes specific to either genotype. Additionally, genes encoding outer membrane proteins (OMPs) specific to genotype 2 M. haemolytica were identified, and the diversity of their protein isoforms was characterized with predominantly unassembled, short-read genomic sequences for up to 1075 additional strains., Results: The pan genome of the 69 sequenced M. haemolytica strains consisted of 3111 genes, of which 1880 comprised a shared core between the genotypes. A core of 112 and 179 genes or gene variants were specific to genotype 1 and 2, respectively. Seven genes encoding predicted OMPs; a peptidase S6, a ligand-gated channel, an autotransporter outer membrane beta-barrel domain-containing protein (AOMB-BD-CP), a porin, and three different trimeric autotransporter adhesins were specific to genotype 2 as their genotype 1 homologs were either pseudogenes, or not detected. The AOMB-BD-CP gene, however, appeared to be truncated across all examined genotype 2 strains and to likely encode dysfunctional protein. Homologous gene sequences from additional M. haemolytica strains confirmed the specificity of the remaining six genotype 2 OMP genes and revealed they encoded low isoform diversity at the population level., Conclusion: Genotype 2 M. haemolytica possess genes encoding conserved OMPs not found intact in more commensally prone genotype 1 strains. Some of the genotype 2 specific genes identified in this study are likely to have important biological roles in the pathogenicity of genotype 2 M. haemolytica, which is the primary bacterial cause of BRD.

Objectives: To identify and analyse the first ESBL gene from Mannheimia haemolytica., Methods: Susceptibility testing was performed according to CLSI. Plasmids were extracted via alkaline lysis and transferred by electrotransformation. The sequence was determined by WGS and confirmed by Sanger sequencing., Results: The M. haemolytica strain 48 showed high cephalosporin MICs. A single plasmid, designated pKKM48, with a size of 4323 bp, was isolated. Plasmid pKKM48 harboured a novel blaROB gene, tentatively designated blaROB-2, and was transferred to Pasteurella multocida B130 and to Escherichia coli JM107. PCR assays and susceptibility testing confirmed the presence and activity of the blaROB-2 gene in the P. multocida and in the E. coli recipient carrying plasmid pKKM48. The transformants had high MICs of all β-lactam antibiotics. An ESBL phenotype was seen in the E. coli transformant when applying the CLSI double-disc confirmatory test for E. coli. The blaROB-2 gene from plasmid pKKM48 differed in three positions from blaROB-1, resulting in two amino acid exchanges and one additional amino acid in the deduced β-lactamase protein. In addition to blaROB-2, pKKM48 harboured mob genes and showed high similarity to other plasmids from Pasteurellaceae., Conclusions: This study described the first ESBL gene in Pasteurellaceae, which may limit the therapeutic options for veterinarians. The transferability to Enterobacteriaceae with the functional activity of the gene in the new host underlines the possibility of the spread of this gene across species or genus boundaries., (© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Standardized definitions for MDR are currently not available in veterinary medicine despite numerous reports indicating that antimicrobial resistance may be increasing among clinically significant bacteria in livestock and companion animals. As such, assessments of MDR presented in veterinary scientific reports are inconsistent. Herein, we apply previously standardized definitions for MDR, XDR and pandrug resistance (PDR) used in human medicine to animal pathogens and veterinary antimicrobial agents in which MDR is defined as an isolate that is not susceptible to at least one agent in at least three antimicrobial classes, XDR is defined as an isolate that is not susceptible to at least one agent in all but one or two available classes and PDR is defined as an isolate that is not susceptible to all agents in all available classes. These definitions may be applied to antimicrobial agents used to treat bovine respiratory disease (BRD) caused by Mannheimia haemolytica, Pasteurella multocida and Histophilus somni and swine respiratory disease (SRD) caused by Actinobacillus pleuropneumoniae, P. multocida and Streptococcus suis, as well as antimicrobial agents used to treat canine skin and soft tissue infections (SSTIs) caused by Staphylococcus and Streptococcus species. Application of these definitions in veterinary medicine should be considered static, whereas the classification of a particular resistance phenotype as MDR, XDR or PDR could change over time as more veterinary-specific clinical breakpoints or antimicrobial classes and/or agents become available in the future.

Antimicrobial susceptibility testing is an essential tool to the veterinarian for selecting the most appropriate agent for treatment of bacterial diseases of animals. The availability of well-defined methods that incorporate the necessary quality controls coupled to clinical outcome data is foundational in providing relevant test results for clinical decisions. Since 1993, the Clinical Laboratory and Standards Institute (CLSI) Subcommittee on Veterinary Antimicrobial Susceptibility Testing (VAST) has developed specific test methods and interpretive criteria for veterinary pathogens. This information has allowed for veterinarians to more effectively treat animal diseases thereby protecting both animal welfare and human food security. Moreover, the availability of standardized test methods for veterinary pathogens has allowed for the development of antimicrobial surveillance programs to detect the emergence of resistance among veterinary pathogens. Future work by the VAST and other groups will be critical to expanding the current test methods and interpretive criteria to more pathogen-antibacterial combinations, as well as, the incorporation of genomic information for routine antimicrobial susceptibility testing in the veterinary diagnostic laboratory.

Danofloxacin is a fluoroquinolone antibacterial agent approved for use in veterinary medicine to treat and control bovine respiratory disease caused by Mannheimia haemolytica or Pasteurella multocida. Susceptible minimal inhibitory concentration (MIC) breakpoint (≤0.25 µg/mL) and disk diffusion interpretive criteria (≥22 mm) values for danofloxacin against M. haemolytica and P. multocida were first approved by the Clinical and Laboratory Standards Institute (CLSI) in 2003. However, intermediate and resistant breakpoint values were not established because only susceptible wild-type populations were evident at the time of breakpoint approvals. Since then, nonsusceptible isolates of M. haemolytica and P. multocida have been identified. We report danofloxacin intermediate MIC breakpoint (0.5 µg/mL) and disk diffusion interpretive criteria (18-21 mm), as well as danofloxacin-resistant MIC breakpoint (≥1 µg/mL) and disk diffusion interpretive criteria (≤17 mm), based on scattergram plots of MIC values versus disk zone diameters and calculated error-bound rates using M. haemolytica and P. multocida isolates recovered from bovine respiratory disease in North America in 2004-2014. These newly established intermediate and resistant clinical breakpoint values have been endorsed by CLSI and can be used for interpreting results from antibacterial susceptibility testing of danofloxacin against M. haemolytica and P. multocida isolated from bovine respiratory disease.

Background: Mannheimia haemolytica typically resides in cattle as a commensal member of the upper respiratory tract microbiome. However, some strains can invade their lungs and cause respiratory disease and death, including those with multi-drug resistance. A nucleotide polymorphism typing system was developed for M. haemolytica from the genome sequences of 1133 North American isolates, and used to identify genetic differences between isolates from the lungs and upper respiratory tract of cattle with and without clinical signs of respiratory disease., Results: A total of 26,081 nucleotide polymorphisms were characterized after quality control filtering of 48,403 putative polymorphisms. Phylogenetic analyses of nucleotide polymorphism genotypes split M. haemolytica into two major genotypes (1 and 2) that each were further divided into multiple subtypes. Multiple polymorphisms were identified with alleles that tagged genotypes 1 or 2, and their respective subtypes. Only genotype 2 M. haemolytica associated with the lungs of diseased cattle and the sequence of a particular integrative and conjugative element (ICE). Additionally, isolates belonging to one subtype of genotype 2 (2b), had the majority of antibiotic resistance genes detected in this study, which were assorted into seven combinations that ranged from 1 to 12 resistance genes., Conclusions: Typing of diverse M. haemolytica by nucleotide polymorphism genotypes successfully identified associations with diseased cattle lungs, ICE sequence, and antibiotic resistance genes. Management of cattle by their carriage of M. haemolytica could be an effective intervention strategy to reduce the prevalence of respiratory disease and supplemental needs for antibiotic treatments in North American herds.

Objectives: The aim of this study was to identify and analyse the first integrative and conjugative element (ICE) from Mannheimia haemolytica, the major bacterial component of the bovine respiratory disease (BRD) complex., Methods: The novel ICEMh1 was discovered in the whole-genome sequence of M. haemolytica 42548 by sequence analysis and comparative genomics. Transfer of ICEMh1 was confirmed by conjugation into Pasteurella multocida recipient cells., Results: ICEMh1 has a size of 92,345 bp and harbours 107 genes. It integrates into a chromosomal tRNA(Leu) copy. Within two resistance gene regions of ∼ 7.4 and 3.3 kb, ICEMh1 harbours five genes, which confer resistance to streptomycin (strA and strB), kanamycin/neomycin (aphA1), tetracycline [tetR-tet(H)] and sulphonamides (sul2). ICEMh1 is related to the recently described ICEPmu1 and both ICEs seem to have evolved from a common ancestor. A region of ICEMh1 that is absent in ICEPmu1 was found in putative ICE regions of other M. haemolytica genomes, suggesting a recombination event between two ICEs. ICEMh1 transfers to P. multocida by conjugation, in which it also uses a tRNA(Leu) as the integration site. PCR assays and susceptibility testing confirmed the presence and activity of the ICEMh1-associated resistance genes in the P. multocida recipient., Conclusions: These findings showed that ICEs, with structurally variable resistance gene regions, are present in BRD-associated Pasteurellaceae, can easily spread across genus borders and enable the acquisition of multidrug resistance via a single horizontal gene transfer event. This poses a threat to efficient antimicrobial chemotherapy of BRD-associated bacterial pathogens., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Clinafloxacin is a broad-spectrum fluoroquinolone that was originally developed and subsequently abandoned in the late 1990s as a human health antibiotic for respiratory diseases. The purpose of this study was to investigate the activity of clinafloxacin as a possible treatment for respiratory disease in cattle and pigs. Minimum inhibitory concentration (MIC) values were determined using Clinical and Laboratory Standards Institute recommended procedures with recent strains from the Zoetis culture collection. Rodent efficacy was determined in CD-1 mice infected systemically or intranasally with bovine Mannheimia haemolytica or Pasteurella multocida, or swine Actinobacillus pleuropneumoniae, and administered clinafloxacin for determination of ED50 (efficacious dose-50%) values. The MIC90 values for clinafloxacin against bovine P. multocida, M. haemolytica, Histophilus somni, and M. bovis were 0.125, 0.5, 0.125, and 1 μg/ml, respectively, and the MIC90 values against swine P. multocida, A. pleuropneumoniae, S. suis, and M. hyopneumoniae were í0.03, í0.03, 0.125, and í0.008 μg/ml, respectively. Efficacy in mouse models showed average ED50 values of 0.019 mg/kg/dose in the bovine M. haemolytica systemic infection model, 0.55 mg/kg in the bovine P. multocida intranasal lung challenge model, 0.08 mg/kg/dose in the bovine P. multocida systemic infection model, and 0.7 mg/kg/dose in the swine A. pleuropneumoniae systemic infection model. Clinafloxacin shows good in vitro activity and efficacy in mouse models and may be a novel treatment alternative for the treatment of respiratory disease in cattle and pigs.

Mannheimia haemolytica is the major bacterial component in the bovine respiratory disease complex, which accounts for considerable economic losses to the cattle industry worldwide. The complete genome sequence of M. haemolytica strain 42548 was determined. It has a size of 2.73 Mb and contains 2,888 genes, including several antibiotic resistance genes.

Background: In recent years, multiresistant Pasteurella multocida isolates from bovine respiratory tract infections have been identified. These isolates have exhibited resistance to most classes of antimicrobial agents commonly used in veterinary medicine, the genetic basis of which, however, is largely unknown., Methods: Genomic DNA of a representative P. multocida isolate was subjected to whole genome sequencing. Genes have been predicted by the YACOP program, compared with the SWISSProt/EMBL databases and manually curated using the annotation software ERGO. Susceptibility testing was performed by broth microdilution according to CLSI recommendations., Results: The analysis of one representative P. multocida isolate identified an 82 kb integrative and conjugative element (ICE) integrated into the chromosomal DNA. This ICE, designated ICEPmu1, harboured 11 resistance genes, which confer resistance to streptomycin/spectinomycin (aadA25), streptomycin (strA and strB), gentamicin (aadB), kanamycin/neomycin (aphA1), tetracycline [tetR-tet(H)], chloramphenicol/florfenicol (floR), sulphonamides (sul2), tilmicosin/clindamycin [erm(42)] or tilmicosin/tulathromycin [msr(E)-mph(E)]. In addition, a complete bla(OXA-2) gene was detected, which, however, appeared to be functionally inactive in P. multocida. These resistance genes were organized in two regions of approximately 15.7 and 9.8 kb. Based on the sequences obtained, it is likely that plasmids, gene cassettes and insertion sequences have played a role in the development of the two resistance gene regions within this ICE., Conclusions: The observation that 12 resistance genes, organized in two resistance gene regions, represent part of an ICE in P. multocida underlines the risk of simultaneous acquisition of multiple resistance genes via a single horizontal gene transfer event.

Background: Integrative and conjugative elements (ICEs) have not been detected in Pasteurella multocida. In this study the multiresistance ICEPmu1 from bovine P. multocida was analysed for its core genes and its ability to conjugatively transfer into strains of the same and different genera., Methods: ICEPmu1 was identified during whole genome sequencing. Coding sequences were predicted by bioinformatic tools and manually curated using the annotation software ERGO. Conjugation into P. multocida, Mannheimia haemolytica and Escherichia coli recipients was performed by mating assays. The presence of ICEPmu1 and its circular intermediate in the recipient strains was confirmed by PCR and sequence analysis. Integration sites were sequenced. Susceptibility testing of the ICEPmu1-carrying recipients was conducted by broth microdilution., Results: The 82 214 bp ICEPmu1 harbours 88 genes. The core genes of ICEPmu1, which are involved in excision/integration and conjugative transfer, resemble those found in a 66 641 bp ICE from Histophilus somni. ICEPmu1 integrates into a tRNA(Leu) and is flanked by 13 bp direct repeats. It is able to conjugatively transfer to P. multocida, M. haemolytica and E. coli, where it also uses a tRNA(Leu) for integration and produces closely related 13 bp direct repeats. PCR assays and susceptibility testing confirmed the presence and the functional activity of the ICEPmu1-associated resistance genes in the recipient strains., Conclusions: The observation that the multiresistance ICEPmu1 is present in a bovine P. multocida and can easily spread across strain and genus boundaries underlines the risk of a rapid dissemination of multiple resistance genes, which will distinctly decrease the therapeutic options.

The mechanism of macrolide-triamilide resistance in Pasteurella multocida has been unknown. During whole-genome sequencing of a multiresistant bovine P. multocida isolate, three new resistance genes, the rRNA methylase gene erm(42), the macrolide transporter gene msr(E), and the macrolide phosphotransferase gene mph(E), were detected. The three genes were PCR amplified, cloned into suitable plasmid vectors, and shown to confer either macrolide-lincosamide resistance [erm(42)] or macrolide-triamilide resistance [msr(E)-mph(E)] in macrolide-susceptible Escherichia coli and P. multocida hosts.

The introduction of newer antimicrobial agents over the past two decades has dramatically improved the treatment of bovine respiratory disease (BRD). In the same time period, the implementation of standardized susceptibility test methods and BRD-specific interpretive criteria has substantially improved the ability to detect clinical resistance in the BRD pathogens. Although overall levels of resistance to the newer antimicrobial agents are generally low, recent data have indicated the potential for emergence and dissemination of a resistant clone in cattle. These data indicate the need for long-term surveillance of antimicrobial resistance in the BRD pathogens and a better understanding of the epidemiology of antimicrobial resistance in these pathogens., (Copyright 2010 Elsevier Inc. All rights reserved.)

The discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents has been described in a recent series of papers. This paper describes the discovery of 1-acylindazol-3-ols as a novel bioisostere of an anthranilic acid. The synthesis and structure-activity relationships of the indazol bioisosteres are described herein.

The purpose of this study was to determine the activities of two antibacterial agents used in the treatment of bovine respiratory infections-tulathromycin, a macrolide, and ceftiofur, a third-generation cephalosporin-alone, in combination with each other, and in combination with each of seven additional antibiotics (tilmicosin, florfenicol, enrofloxacin, danofloxacin, ampicillin, tetracycline, and penicillin G) against bovine Pasteurella multocida (n = 60) and Mannheimia haemolytica (n = 10) isolates for determination of synergy, antagonism, or indifference. Of 458 organism-drug combinations, 160 combinations of tulathromycin and 209 combinations of ceftiofur with eight antimicrobial drugs were indifferent. One combination was antagonistic (ceftiofur + florfenicol against one isolate of P. multocida). Time-kill studies showed loss of cidality for ceftiofur when combined with florfenicol at 1x the minimal inhibitory concentration. Overall, the in vitro data demonstrated that tulathromycin and ceftiofur, in combination with each other or seven other antimicrobial agents, primarily produce an indifferent response with no occurrences of synergism and rare occurrences of antagonism.

QPT-1 was discovered in a compound library by high-throughput screening and triage for substances with whole-cell antibacterial activity. This totally synthetic compound is an unusual barbituric acid derivative whose activity resides in the (-)-enantiomer. QPT-1 had activity against a broad spectrum of pathogenic, antibiotic-resistant bacteria, was nontoxic to eukaryotic cells, and showed oral efficacy in a murine infection model, all before any medicinal chemistry optimization. Biochemical and genetic characterization showed that the QPT-1 targets the beta subunit of bacterial type II topoisomerases via a mechanism of inhibition distinct from the mechanisms of fluoroquinolones and novobiocin. Given these attributes, this compound represents a promising new class of antibacterial agents. The success of this reverse genomics effort demonstrates the utility of exploring strategies that are alternatives to target-based screens in antibacterial drug discovery.

Background: We report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features., Results: We have produced a genotyping microarray for rice, comprising 880 single feature polymorphism (SFP) elements derived from insertions/deletions identified by aligning genomic sequences of the japonica cultivar Nipponbare and the indica cultivar 93-11. The SFPs were experimentally verified by hybridization with labeled genomic DNA prepared from the two cultivars. Using the genotyping microarrays, we found high levels of polymorphism across diverse rice accessions, and were able to classify all five subpopulations of rice with high bootstrap support. The microarrays were used for mapping of a gene conferring resistance to Magnaporthe grisea, the causative organism of rice blast disease, by quantitative genotyping of samples from a recombinant inbred line population pooled by phenotype., Conclusion: We anticipate this microarray-based genotyping platform, based on its low cost-per-sample, to be particularly useful in applications requiring whole-genome molecular marker coverage across large numbers of individuals.

Infections caused by multidrug-resistant Gram-negative pathogens play a major role in the morbidity and mortality of hospitalized patients. The rise of resistance to current antibiotic therapies has made the discovery of new agents urgent. One of the major antibiotic resistance mechanisms utilized by more than 15 species of Gram-negative bacterial cells is the Resistance Nodulation Division (RND) efflux pump, which eliminates several classes of antibiotics such as penicillins and cephalosporin macrolides aminoglycosides, fluoroquinolonesx and tetracyclines. Here we describe a multistep process to identify compounds that inhibit the RND-type efflux pumps. This involves measuring the inhibition of accumulation of ethidium bromide in E. coli or Haemophilus influenzae cells and confirming that the inhibition is specific for the efflux pumps by using genetic constructs and biochemical methods to measure nonspecific inhibition due to e.g. intrinsic antibacterial activity or membrane disruption. In whole bacterial cells synergism antagonism or indifference of the combination of an antibiotic with the putative inhibitor is determined and this is then confirmed by quantitating viable bacterial cells in liquid culture over 24 h.

The history of rice domestication has long been a subject of debate. Recently obtained genetic evidence provides new insights into this complex story. Genome-wide studies of variation demonstrate that the two varietal groups in Oryza sativa (indica and japonica) arose from genetically distinct gene pools within a common wild ancestor, Oryza rufipogon, suggesting multiple domestications of O. sativa. However, the evolutionary history of recently cloned domestication genes adds another layer of complexity to the domestication of rice. Although some alleles exist only within specific subpopulations, as would be expected if the domestications occurred independently, other major domestication alleles are common to all cultivated O. sativa varieties. Our current view of rice domestication supports multiple domestications coupled with limited introgression that transferred key domestication alleles between divergent rice gene pools.

Previously we reported the discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents which suffered from extensive protein binding. This report describes efforts directed toward understanding the relationship of the acidity of the carboxylic acid with the extent of protein binding. The pK(a) of the acid was modified via the synthesis of a number of anthranilic acid analogs which vary the aromatic ring substituent at the 4-position. The pK(a) and HSA binding constants have been determined for each of the analogs. Our results indicate a correlation between pK(a) and HSA K(d). The physical properties and antibacterial activities will be discussed as well as how these results help address the protein binding issue with this series of compounds.

Here we report that the change from the red seeds of wild rice to the white seeds of cultivated rice (Oryza sativa) resulted from the strong selective sweep of a single mutation, a frame-shift deletion within the Rc gene that is found in 97.9% of white rice varieties today. A second mutation, also within Rc, is present in less than 3% of white accessions surveyed. Haplotype analysis revealed that the predominant mutation originated in the japonica subspecies and crossed both geographic and sterility barriers to move into the indica subspecies. A little less than one Mb of japonica DNA hitchhiked with the rc allele into most indica varieties, suggesting that other linked domestication alleles may have been transferred from japonica to indica along with white pericarp color. Our finding provides evidence of active cultural exchange among ancient farmers over the course of rice domestication coupled with very strong, positive selection for a single white allele in both subspecies of O. sativa., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

The discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents which suffered from extensive protein binding has been previously reported. The structure-activity relationships around the carboxylic acid substituent are described herein. This acid was replaced by several alternative functional groups in attempts to retain bioactivity while reducing protein binding. Only groups with an acidic proton retained activity, and analogs containing those groups maintained the protein binding inherent to this class of antibacterial agents.

In the past few years a significant effort has been devoted by Pharmacia toward the discovery of novel antibiotics. We have recently described the identification of an anthranilic acid lead 1 and the optimization resulting in the advanced lead 2. In this report, we describe the preparation of several selected analogs to probe the dependency of this template for antibacterial activity and the affinity these compounds have for human serum albumin (HSA). These analogs illustrate that decreased affinity for HSA can be achieved while retaining relevant antibacterial activity. The most important factor for reduced HSA affinity is decrease in logP rather than a structural change.

In the past few years, a significant effort has been devoted by Pharmacia toward the discovery of novel antibiotics. We have recently described the identification of an anthranilic acid lead 1 and the optimization resulting in the advanced lead 2. In this report, we describe the preparation of several selected amide bioisosteres connecting the A- and the B-rings. The E-alkene provided a rigid analog with equal potency to the corresponding amide. This indicates that the amide is not a recognition element rather acts as an appropriate spatial linker of the two important aryl A and B rings. The work here clearly demonstrates that the amide linker can be replaced with several functionalities without significant deterioration in the MIC activity.

Discovery of novel antibacterial agents is a significant challenge. We have recently reported on our discovery of novel antibacterial agents in which we have rapidly optimized potency utilizing a parallel chemistry approach. These advanced leads suffer from high affinity for human serum albumin (HSA). In an effort to decrease the affinity for HSA we have prepared a series of heterocyclic analogs, which retained antibacterial activity and demonstrated reduced affinity for HSA.

The novel bacterial transcription/translation (TT) inhibitor 1 was identified through a combination of high throughput screening and exploratory medicinal chemistry. Initial optimization of the anthranilic acid moiety and sulfonamide amine diversity was accomplished via 1- and two-dimensional solution phase libraries, resulting in an improvement in the MIC of the lead from 64 to 8mug/mL (compound 4l). Subsequent modification of the central aromatic ring and further refinement of the sulfonamide amines required the development of a solid phase route on Wang resin. The resulting libraries generated a number of potent antibacterials with MICs of 1mug/mL (e.g., 10b, 12, and 13). During the course of this work, it became apparent that the antibacterial activity of the series is not fully correlated with TT inhibition, suggesting that at least one additional mechanism of action is operative.

Rc is a domestication-related gene required for red pericarp in rice (Oryza sativa). The red grain color is ubiquitous among the wild ancestors of O. sativa, in which it is closely associated with seed shattering and dormancy. Rc encodes a basic helix-loop-helix (bHLH) protein that was fine-mapped to an 18.5-kb region on rice chromosome 7 using a cross between Oryza rufipogon (red pericarp) and O. sativa cv Jefferson (white pericarp). Sequencing of the alleles from both mapping parents as well as from two independent genetic stocks of Rc revealed that the dominant red allele differed from the recessive white allele by a 14-bp deletion within exon 6 that knocked out the bHLH domain of the protein. A premature stop codon was identified in the second mutant stock that had a light red pericarp. RT-PCR experiments confirmed that the Rc gene was expressed in both red- and white-grained rice but that a shortened transcript was present in white varieties. Phylogenetic analysis, supported by comparative mapping in rice and maize (Zea mays), showed that Rc, a positive regulator of proanthocyanidin, is orthologous with INTENSIFIER1, a negative regulator of anthocyanin production in maize, and is not in the same clade as rice bHLH anthocyanin regulators.

Screening of our compound collection identified PNU-92560, a 2-[1,3,4]thiadiazolo[3,2-a]pyrimidine-6-carboxamide, as a novel antibacterial agent. Extensive analogue development identified that the 2-position of the thiadiazole could be functionalized with a linker that would allow the compound to be attached to a solid support. The extreme insolubility of the analogues prevented the mechanism of action for these compounds to be determined utilizing traditional methodology. The solid-supported compounds were utilized as affinity columns to identify elongation factor Tu (EF-Tu) as a putative target for this class of compounds. The activity of the compounds in a metabolic labeling experiments and in translation assay supports the identity of the target for these compounds to be EF-Tu.

The activities of linezolid, an oxazolidinone antibacterial agent active against gram-positive organisms, alone and in combination with 35 antimicrobial agents were tested in vitro against methicillin-sensitive (n = 1 to 2 strains) and methicillin-resistant (n = 8 to 10) Staphylococcus aureus strains; vancomycin-sensitive (n = 6) and vancomycin-resistant (n = 6 to 8) Enterococcus faecalis strains; vancomycin-sensitive (n = 5) and vancomycin-resistant (n = 6) Enterococcus faecium strains; penicillin-sensitive (n = 2 to 5), penicillin-intermediate (n = 5 to 6), and penicillin-resistant (n = 5 to 6) Streptococcus pneumoniae strains; Escherichia coli (n = 6); and Klebsiella pneumoniae (n = 6). The fractional inhibitory concentration indices of linezolid in combination with other antimicrobial agents for the organisms tested were generated on checkerboard broth microdilution plates prepared by a semiautomated method. Of 1,380 organism-drug combinations, 1,369 (99.2%) combinations of linezolid with 28 antimicrobial drugs were indifferent, 9 combinations (0.65%) of linezolid with 6 drugs (amoxicillin, erythromycin, imipenem, sparfloxacin, teicoplanin, and tetracycline) were synergistic, and 2 combinations (0.15%) of linezolid with 2 drugs (ofloxacin and sparfloxacin) were antagonistic. Overall, the in vitro data demonstrated that linezolid combined with other antimicrobial agents primarily produces an indifferent response, with infrequent occurrences of synergism and antagonism.

In a chemical mutagenesis screen, we identified two zebrafish mutants that possessed small pupils. Genetic complementation revealed these two lines are due to mutations in different genes. The phenotypes of the two mutants were characterized using histologic, immunohistochemical, and tissue transplantation techniques. The arrested lens (arl) mutant exhibits a small eye and pupil phenotype at 48 hr postfertilization (hpf) and lacks any histologically identifiable lens structures by 5 days postfertilization (dpf). In contrast, the disrupted lens (dsl) mutants are phenotypically normal until 5 dpf, and then undergo lens disorganization and cell degeneration that is apparent by 7 dpf. Histology reveals the arl mutant terminates lens cell differentiation by 48 hpf, whereas the dsl lens exhibits a defective lens epithelial cell population at 5 dpf. Lens transplantation experiments demonstrate both mutations are autonomous to the lens tissue. Immunohistochemistry reveals the retinal cells may suffer subtle effects, possibly due to the lens abnormalities., (Copyright 2001 Wiley-Liss, Inc.)

This paper describes the discovery of alpha-trifluoroketoacetamides as potent antibacterial agents against Gram-positive organisms. The initial SAR indicates that the aryl ethyl side chain is essential in maintaining antibacterial activity. The SAR observations have been utilized to design a bioisostere for the alpha-trifluoroketoacetamide with good activity against Gram-positive organisms.

Important resistance patterns in Gram-negative pathogens include active efflux of antibiotics out of the cell via a cellular pump and decreased membrane permeability. A 3-arylpiperidine derivative (1) has been identified by high-throughput assay as a potentiator with an IC(50) approximately 90 microM. This report details the evaluation of the tether length, aryl substitution and the importance of the fluorine on antibiotic accumulation. Evaluation of various tether lengths demonstrated that the two-carbon tethered analogues are optimal. Removal of the fluorine has a modest effect on antibiotic accumulation and the defluorinated analogue 17 is equally potent to the original lead 1.

Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococcus aureus PDF afforded a very potent inhibitor with an IC(50) in the low nanomolar range. Unfortunately, the compound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF and antibacterial activity.

An open, non-comparative study of 10 weeks' duration was conducted in general practice to assess the safety of amlodipine in patients with mild to moderate hypertension. Of the 5352 patients entering the study, 5135 received amlodipine; 4621 patients (90%) with a mean age of 58.2 years completed the study. Normalisation of blood pressure was achieved in over 80% of patients with a mean reduction of 21/15 mmHg. The mean final dose of amlodipine was 6.8 mg/day. Adverse experiences possibly related to amlodipine were reported by 19.3% of patients, and overall adverse events led to withdrawal in 6.7% of patients. The most common reported side-effect was oedema. The frequency of headache was almost identical in older and younger patients and oedema, flushing and dizziness were seen only slightly more often in elderly patients. Ninety per cent of patients were considered by their GP to have shown excellent or good toleration of therapy. Over 85% of patients elected to continue on amlodipine therapy after completion of the study.