In vitro and in vivo activity of GT-1, a novel siderophore cephalosporin, and GT-055, a broad-spectrum ß-lactamase inhibitor, against biothreat and ESKAPE pathogens.

Antimicrobial-resistance (AMR) has become an increasingly difficult issue to overcome for bacteria associated with both community- and hospital-acquired infections as well as potential biodefense threats. The need to identify new therapeutics of novel classes and/or with unique mechanisms is critical to combatting AMR in the coming years. GT-1 (LCB10-0200), a siderophore-linked cephalosporin, is one such novel option and is formulated to be used either alone or in combination with a novel broad-spectrum ß-lactamase inhibitor, GT-055 (LCB18-055). This study assessed the in vitro and in vivo efficacy of GT-1 and GT-055 against a broad array of multi-drug resistant and biothreat pathogens. Here, we demonstrated sub-4 µg ml-1 efficacy against a number of pathogens in vitro. We further determined that in mice infected via aerosol route with Yersinia pestis, efficacy of GT-1/GT-055 treatment is at least equivalent to the comparator antibiotic, ciprofloxacin.

Antibiotic Therapy of Plague: A Review.

Plague-a deadly disease caused by the bacterium Yersinia pestis-is still an international public health concern. There are three main clinical forms: bubonic plague, septicemic plague, and pulmonary plague. In all three forms, the symptoms appear suddenly and progress very rapidly. Early antibiotic therapy is essential for countering the disease. Several classes of antibiotics (e.g., tetracyclines, fluoroquinolones, aminoglycosides, sulfonamides, chloramphenicol, rifamycin, and ß-lactams) are active in vitro against the majority of Y. pestis strains and have demonstrated efficacy in various animal models. However, some discrepancies have been reported. Hence, health authorities have approved and recommended several drugs for prophylactic or curative use. Only monotherapy is currently recommended; combination therapy has not shown any benefits in preclinical studies or case reports. Concerns about the emergence of multidrug-resistant strains of Y. pestis have led to the development of new classes of antibiotics and other therapeutics (e.g., LpxC inhibitors, cationic peptides, antivirulence drugs, predatory bacteria, phages, immunotherapy, host-directed therapy, and nutritional immunity). It is difficult to know which of the currently available treatments or therapeutics in development will be most effective for a given form of plague. This is due to the lack of standardization in preclinical studies, conflicting data from case reports, and the small number of clinical trials performed to date.

Novel Disk Diffusion Assay on Magnesium Oxalate Agar To Evaluate the Susceptibility of Yersinia pestis to Type III Secretion System Inhibitors.

Current methods for screening small molecules that inhibit the plasmid pCD1-encoded Yersinia pestis type III secretion system (T3SS) include lengthy growth curves followed by multistep luminescence assays or Western blot assays to detect secretion, or lack thereof, of effector proteins. The goal of this research was to develop a novel disk diffusion assay on magnesium oxalate (MOX) agar as a simple way to evaluate the susceptibility of Y. pestis to type III secretion system inhibitors. MOX agar produces distinct Y. pestis growth characteristics based on the bacteria's ability or inability to secrete effector proteins; small, barely visible colonies are observed when secretion is activated versus larger, readily visible colonies when secretion is inhibited. Wild-type Y. pestis was diluted and spread onto a MOX agar plate. Disks containing 20 µl of various concentrations of imidocarb dipropionate, a known Y. pestis T3SS inhibitor, or distilled water (dH2O) were placed on the plate. After incubation at 37°C for 48 h, visible colonies of Y. pestis were observed surrounding the disks with imidocarb dipropionate, suggesting that T3S was inhibited. The diameter of the growth of colonies surrounding the disks increased as the concentration of the T3SS inhibitor increased. Imidocarb dipropionate was also able to inhibit Y. pestis strains lacking effector Yops and Yop chaperones, suggesting that they are not necessary for T3S inhibition. This disk diffusion assay is a feasible and useful method for testing the susceptibility of Y. pestis to type III secretion system inhibitors and has the potential to be used in a clinical setting. IMPORTANCE Disk diffusion assays have traditionally been used as a simple and effective way to screen compounds for antibacterial activity and to determine the susceptibility of pathogens to antibiotics; however, they are limited to detecting growth inhibition only. Consequently, antimicrobial agents that inhibit virulence factors, but not growth, would not be detected. Therefore, we developed a disk diffusion assay that could detect inhibition of bacterial virulence factors, specifically, type III secretion systems (T3SSs), needle-like structures used by several pathogenic bacteria to inject host cells with effector proteins and cause disease. We demonstrate that magnesium oxalate (MOX) agar can be used in a disk diffusion assay to detect inhibition of the T3SS of Yersinia pestis, the causative agent of bubonic plague, by small-molecule inhibitors. This assay may be useful for screening additional small molecules that target bacterial T3SSs or testing the susceptibility of patient-derived samples to drugs that target T3SSs.

A novel mechanism of streptomycin resistance in Yersinia pestis: Mutation in the rpsL gene.

Streptomycin is considered to be one of the effective antibiotics for the treatment of plague. In order to investigate the streptomycin resistance of Y. pestis in China, we evaluated streptomycin susceptibility of 536 Y. pestis strains in China in vitro using the minimal inhibitory concentration (MIC) and screened streptomycin resistance-associated genes (strA and strB) by PCR method. A clinical Y. pestis isolate (S19960127) exhibited high-level resistance to streptomycin (the MIC was 4,096 mg/L). The strain (biovar antiqua) was isolated from a pneumonic plague outbreak in 1996 in Tibet Autonomous Region, China, belonging to the Marmota himalayana Qinghai-Tibet Plateau plague focus. In contrast to previously reported streptomycin resistance mediated by conjugative plasmids, the genome sequencing and allelic replacement experiments demonstrated that an rpsL gene (ribosomal protein S12) mutation with substitution of amino-acid 43 (K43R) was responsible for the high-level resistance to streptomycin in strain S19960127, which is consistent with the mutation reported in some streptomycin-resistant Mycobacterium tuberculosis strains. Streptomycin is used as the first-line treatment against plague in many countries. The emergence of streptomycin resistance in Y. pestis represents a critical public health problem. So streptomycin susceptibility monitoring of Y. pestis isolates should not only include plasmid-mediated resistance but also include the ribosomal protein S12 gene (rpsL) mutation, especially when treatment failure is suspected due to antibiotic resistance.

VOC fingerprints: metabolomic signatures of biothreat agents with and without antibiotic resistance.

Category A and B biothreat agents are deemed to be of great concern by the US Centers for Disease Control and Prevention (CDC) and include the bacteria Francisella tularensis, Yersinia pestis, Burkholderia mallei, and Brucella species. Underscored by the impact of the 2020 SARS-CoV-2 outbreak, 2016 Zika pandemic, 2014 Ebola outbreak, 2001 anthrax letter attacks, and 1984 Rajneeshee Salmonella attacks, the threat of future epidemics/pandemics and/or terrorist/criminal use of pathogenic organisms warrants continued exploration and development of both classic and alternative methods of detecting biothreat agents. Volatile organic compounds (VOCs) comprise a large and highly diverse group of carbon-based molecules, generally related by their volatility at ambient temperature. Recently, the diagnostic potential of VOCs has been realized, as correlations between the microbial VOC metabolome and specific bacterial pathogens have been identified. Herein, we describe the use of microbial VOC profiles as fingerprints for the identification of biothreat-relevant microbes, and for differentiating between a kanamycin susceptible and resistant strain. Additionally, we demonstrate microbial VOC profiling using a rapid-throughput VOC metabolomics method we refer to as 'simultaneous multifiber headspace solid-phase microextraction' (simulti-hSPME). Finally, through VOC analysis, we illustrate a rapid non-invasive approach to the diagnosis of BALB/c mice infected with either F. tularensis SCHU S4 or Y. pestis CO92.

Immunotropic Properties of an Experimental Synthetic Selenium-Organic Compound.

We studied immunotropic properties of synthetic selenium-organic preparation 2,6-dipyridinium-9-selenabicyclo[3.3.1]nonyl dibromide (974zh). The experimental preparation reduced the cAMP/cGMP ratio, which indicated an increase in proliferative activity of cells of immunocompetent organs (thymus and spleen) in experimental animals. It was shown that 974zh intensified the immune response to Yersinia pestis EV thereby increasing the resistance to the plague agent.

Dapsone is not a Pharmacodynamic Lead Compound for its Aryl Derivatives.

BACKGROUND: The relatedness between the linear equations of thermodynamics and QSAR was studied thanks to the recently elucidated crystal structure complexes between sulfonamide pterin conjugates and dihydropteroate synthase (DHPS) together with a published set of thirty- six synthetic dapsone derivatives with their reported entropy-driven activity data. Only a few congeners were slightly better than dapsone. OBJECTIVE: Our study aimed at demonstrating the applicability of thermodynamic QSAR and to shed light on the mechanistic aspects of sulfone binding to DHPS. METHODS: To this end ligand docking to DHPS, quantum mechanical properties, 2D- and 3D-QSAR as well as Principle Component Analysis (PCA) were carried out. RESULTS: The short aryl substituents of the docked pterin-sulfa conjugates were outward oriented into the solvent space without interacting with target residues which explains why binding enthalpy (ΔH) did not correlate with potency. PCA revealed how chemically informative descriptors are evenly loaded on the first three PCs (interpreted as ΔG, ΔH and ΔS), while chemically cryptic ones reflected higher dimensional (complex) loadings. CONCLUSION: It is safe to utter that synthesis efforts to introduce short side chains for aryl derivatization of the dapsone scaffold have failed in the past. On theoretical grounds we provide computed evidence why dapsone is not a pharmacodynamic lead for drug profiling because enthalpic terms do not change significantly at the moment of ligand binding to target.

Mutually constructive roles of Ail and LPS in Yersinia pestis serum survival.

The outer membrane is a key virulence determinant of gram-negative bacteria. In Yersinia pestis, the deadly agent that causes plague, the protein Ail and lipopolysaccharide (LPS)6 enhance lethality by promoting resistance to human innate immunity and antibiotics, enabling bacteria to proliferate in the human host. Their functions are highly coordinated. Here we describe how they cooperate to promote pathogenesis. Using a multidisciplinary approach, we identify mutually constructive interactions between Ail and LPS that produce an extended conformation of Ail at the membrane surface, cause thickening and rigidification of the LPS membrane, and collectively promote Y. pestis survival in human serum, antibiotic resistance, and cell envelope integrity. The results highlight the importance of the Ail-LPS assembly as an organized whole, rather than its individual components, and provide a handle for targeting Y. pestis pathogenesis.

Activation of Heme Oxygenase Expression by Cobalt Protoporphyrin Treatment Prevents Pneumonic Plague Caused by Inhalation of Yersinia pestis.

Pneumonic plague, caused by the Gram-negative bacteria Yersinia pestis, is an invasive, rapidly progressing disease with poor survival rates. Following inhalation of Y. pestis, bacterial invasion of the lungs and a tissue-damaging inflammatory response allows vascular spread of the infection. Consequently, primary pneumonic plague is a multiorgan disease involving sepsis and necrosis of immune tissues and the liver, as well as bronchopneumonia and rampant bacterial growth. Given the likely role of the hyperinflammatory response in accelerating the destruction of tissue, in this work we evaluated the therapeutic potential of the inducible cytoprotective enzyme heme oxygenase 1 (HO-1) against primary pneumonic plague. On its own, the HO-1 inducer cobalt protoporphyrin IX (CoPP) provided mice protection from lethal challenge with Y. pestis CO92 with improved pulmonary bacterial clearance and a dampened inflammatory response compared to vehicle-treated mice. Furthermore, CoPP treatment combined with doxycycline strongly enhanced protection in a rat aerosol challenge model. Compared to doxycycline alone, CoPP treatment increased survival, with a 3-log decrease in median bacterial titer recovered from the lungs and the general absence of a systemic hyperinflammatory response. In contrast, treatment with the HO-1 inhibitor SnPP had no detectable impact on doxycycline efficacy. The combined data indicate that countering inflammatory toxicity by therapeutically inducing HO-1 is effective in reducing the rampant growth of Y. pestis and preventing pneumonic plague.

A Recombinant Attenuated Yersinia pseudotuberculosis Vaccine Delivering a Y. pestis YopENt138-LcrV Fusion Elicits Broad Protection against Plague and Yersiniosis in Mice.

In this study, a novel recombinant attenuated Yersinia pseudotuberculosis PB1+ strain (χ10069) engineered with ΔyopK ΔyopJ Δasd triple mutations was used to deliver a Y. pestis fusion protein, YopE amino acid 1 to 138-LcrV (YopENt138-LcrV), to Swiss Webster mice as a protective antigen against infections by yersiniae. χ10069 bacteria harboring the pYA5199 plasmid constitutively synthesized the YopENt138-LcrV fusion protein and secreted it via the type 3 secretion system (T3SS) at 37°C under calcium-deprived conditions. The attenuated strain χ10069(pYA5199) was manifested by the establishment of controlled infection in different tissues without developing conspicuous signs of disease in histopathological analysis of microtome sections. A single-dose oral immunization of χ10069(pYA5199) induced strong serum antibody titers (log10 mean value, 4.2), secretory IgA in bronchoalveolar lavage (BAL) fluid from immunized mice, and Yersinia-specific CD4+ and CD8+ T cells producing high levels of tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin 2 (IL-2), as well as IL-17, in both lungs and spleens of immunized mice, conferring comprehensive Th1- and Th2-mediated immune responses and protection against bubonic and pneumonic plague challenges, with 80% and 90% survival, respectively. Mice immunized with χ10069(pYA5199) also exhibited complete protection against lethal oral infections by Yersinia enterocolitica WA and Y. pseudotuberculosis PB1+. These findings indicated that χ10069(pYA5199) as an oral vaccine induces protective immunity to prevent bubonic and pneumonic plague, as well as yersiniosis, in mice and would be a promising oral vaccine candidate for protection against plague and yersiniosis for human and veterinary applications.

Characterization of a Transposon Tn5-Generated Mutant of Yersinia pestis Defective in Lipooligosaccharide Biosynthesis.

To identify Yersinia pestis genes involved in the microbe's resistance to cationic antimicrobial peptides, the strategy of random transposon mutagenesis with a Tn5 minitransposon was used, and the library was screened for detecting polymyxin B (PMB) susceptible mutants. The mutation responsible for PMB-sensitive phenotype and the lipopolysaccharide (LPS) structure were characterized for the Y. pestis strain KM218-A3. In this strain the mini-Tn5 was located in an open reading frame with the product homologous to the E. coli protein GmhB (82% identity) functioning as d-glycero-d-manno-heptose-1,7-diphosphate phosphatase. ESI FT ICR mass spectrometry of anions was used to study the structure of the unmodified LPS of Y. pestis KM218-A3, and molecules were revealed with the full-size LPS core or with two types of an incomplete core: consisting of Kdo-Kdo or Ko-Kdo disaccharides and Hep-(Kdo)-Kdo or Hep-(Ko)-Kdo trisaccharides. The performed complementation confirmed that the defect in the biological properties of the mutant strain was caused by inactivation of the gmhB gene. These findings indicated that the gmhB gene product of Y. pestis is essential for production of wild-type LPS resistant to antimicrobial peptides and serum.

Rapid antimicrobial susceptibility testing and ß-lactam-induced cell morphology changes of Gram-negative biological threat pathogens by optical screening.

BACKGROUND: For Yersinia pestis, Burkholderia pseudomallei, and Burkholderia mallei, conventional broth microdilution (BMD) is considered the gold standard for antimicrobial susceptibility testing (AST) and, depending on the species, requires an incubation period of 16-20 h, or 24-48 h according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. After a diagnosis of plague, melioidosis or glanders during an outbreak or after an exposure event, the timely distribution of appropriate antibiotics for treatment or post-exposure prophylaxis of affected populations could reduce mortality rates. RESULTS: Herein, we developed and evaluated a rapid, automated susceptibility test for these Gram-negative bacterial pathogens based on time-lapse imaging of cells incubating in BMD microtitre drug panels using an optical screening instrument (oCelloScope). In real-time, the instrument screened each inoculated well containing broth with various concentrations of antibiotics published by CLSI for primary testing: ciprofloxacin (CIP), doxycycline (DOX) and gentamicin (GEN) for Y. pestis; imipenem (IPM), ceftazidime (CAZ) and DOX for B. mallei; and IPM, DOX, CAZ, amoxicillin-clavulanic acid (AMC) and trimethoprim-sulfamethoxazole (SXT) for B. pseudomallei. Based on automated growth kinetic data, the time required to accurately determine susceptibility decreased by ≥70% for Y. pestis and ≥ 50% for B. mallei and B. pseudomallei compared to the times required for conventional BMD testing. Susceptibility to GEN, IPM and DOX could be determined in as early as three to six hours. In the presence of CAZ, susceptibility based on instrument-derived growth values could not be determined for the majority of B. pseudomallei and B. mallei strains tested. Time-lapse video imaging of these cultures revealed that the formation of filaments in the presence of this cephalosporin at inhibitory concentrations was detected as growth. Other ß-lactam-induced cell morphology changes, such as the formation of spheroplasts and rapid cell lysis, were also observed and appear to be strain- and antibiotic concentration-dependent. CONCLUSIONS: A rapid, functional AST was developed and real-time video footage captured ß-lactam-induced morphologies of wild-type B. mallei and B. pseudomallei strains in broth. Optical screening reduced the time to results required for AST of three Gram-negative biothreat pathogens using clinically relevant, first-line antibiotics compared to conventional BMD.

Rapid Antibiotic Susceptibility Determination for Yersinia pestis Using Flow Cytometry Spectral Intensity Ratio (SIR) Fluorescence Analysis.

Rapid antimicrobial susceptibility tests (ASTs) are essential tool for proper treatment of patients infected by Yersinia pestis (Y. pestis), the causative agent of plague, or for post-exposure prophylaxis of a population exposed to a naturally acquired or deliberately prepared resistant variant. The standard AST of Y. pestis is based on bacterial growth and requires 24-48 h of incubation in addition to the time required for prior isolation of a bacterial culture from the clinical or environmental sample, which may take an additional 24-48 h. In this study, we present a new and rapid AST method based on a fluorescence determination of the minimum inhibitory concentration (MIC). Our method includes the incubation of bacteria with an antibiotic, followed by staining of the bacteria with oxonol dye (SynaptoGreen C4/FM1-43), which enables the rapid detection of an antibiotic's effect on bacterial viability. We show that stained, non-viable bacteria exhibit a spectral redshift and an increase in fluorescence intensity compared to intact control bacteria. Based on these criteria, we developed a rapid flow cytometer measurement procedure and a unique spectral intensity ratio (SIR) analysis that enables determination of antibiotic susceptibility for Y. pestis within 6 h instead of the 24 to 48 h required for the standard AST. This new rapid determination of antibiotic susceptibility could be crucial for reducing mortality and preventing the spread of disease.

Sensitivity of Coriandrum sativum extract on bacterial pathogens isolated from digestive system of rabbits, and its role on in vitro cecal gas production and fermentation.

The present context was aimed to investigate the antibacterial potency of aqueous extract of coriander (Coriandrum sativum L.) leaves against bacterial pathogens isolated from the organs associated with digestive system of rabbit. This study also evaluated the influence of varied doses of aqueous extract of C. sativum (AECS) leaves on in vitro gas production (GP), methane (CH4) production, and some other pivotal fermentation parameters from caecal sample of rabbits. The pathogenic bacteria were isolated from mouth, caecum, and anus of rabbits, and further identified through morphological, biochemical, and molecular tools. The growth inhibitory characteristics of AECS against pathogens were determined using disc diffusion assay. Surprisingly, the result revealed lack of antibacterial potential at tested concentrations. Further, in order to demonstrate the in vitro GP and fermentation parameters in rabbits, four treatments comprising of 0, 0.6, 1.2, and 1.8 mL extract/g dry matter (DM) of AECS were used. Results showed no linear or quadratic effect (P > 0.05) on in vitro GP and CH4 production after the supplementation of AECS in the feeding diet. However, the inclusion of AECS at the concentration of 1.8 mL/g DM exhibited the lowest asymptotic CH4 production and initial delay prior to CH4 production. Similarly, the addition of AECS at 1.8 mL/g DM concentration reduced asymptotic GP as well as CH4 production, and improved fermentation parameters of rabbits when compared with the control and other tested doses. In a nutshell, the tested doses of AECS showed lack of antibacterial trait against the pathogenic bacteria isolated from mouth, caecum, and anus of rabbits. Besides, the AECS exhibited the unique potentiality of reducing GP and improving diversified fermentation parameters in rabbits, thereby suggesting its plausible role as an alternative to commercially available growth promoters in livestock industries.

Mixed pneumonic plague and nosocomial MDR-bacterial infection of lung: a rare case report.

BACKGROUND: Plague is a life-threatening disease caused by the bacterium, Yersinia pestis. Madagascar is the leading country for human plague cases worldwide. Human plague is a serious disease, particularly in its septicaemic and pneumonic forms. We report a case of pneumonic plague co-infected by a MDR-Stenotrophomonas maltophilia. CASE PRESENTATION: A 24 year-old man originated from Soavinandriana, a plague focus, felt uneasy and developed high fever with chills. He started treatment by himself, by private medical care and by a traditional healer for nine days moving several times from place to place. His condition had deteriorated when he presented to a district hospital with a syndrome of dyspnea, bronchial rale and altered state of consciousness. Two days later, plague diagnosis, performed as a last resort, revealed a positive F1 antigen on rapid diagnostic test. Additional tests (pla PCR and plague serology) evidenced a Y. pestis infection. However, streptomycin treatment did not achieve a complete recovery as the course of disease was complicated by the presence of MDR-S. maltophilia in his lung. This opportunistic infection could have been favored by an immunosuppression due to Y. pestis pulmonary infection and probably been acquired during his stay at a District Hospital. He was treated with a combination of ciprofloxacin and gentamycin and recovered fully. CONCLUSIONS: Pneumonic plague infection may promote another virulent or avirulent bacterial infection particularly when it is not initially suspected. However, coinfection is rarely described and its occurrence frequency is unknown. In middle or low resources areas, which is the case of most plague endemic countries, control and prevention of infections in health facilities is not optimal. Co-infection with an opportunistic pathogen agent, such as S. maltophilia, is a risk which must not be disregarded as demonstrated by this case report. When deciding of a national control strategy, it should be taken into account in the choice of the first line treatment.

A Single Amino Acid Change in the Response Regulator PhoP, Acquired during Yersinia pestis Evolution, Affects PhoP Target Gene Transcription and Polymyxin B Susceptibility.

Yersinia pestis, the causative agent of plague, evolved from the closely related pathogen Yersinia pseudotuberculosis During its emergence, Y. pestis is believed to have acquired its unique pathogenic characteristics through numerous gene gains/losses, genomic rearrangements, and single nucleotide polymorphism (SNP) changes. One such SNP creates a single amino acid variation in the DNA binding domain of PhoP, the response regulator in the PhoP/PhoQ two-component system. Y. pseudotuberculosis and the basal human-avirulent strains of Y. pestis harbor glycines at position 215 of PhoP, whereas the modern human-virulent strains (e.g., KIM and CO92) harbor serines at this residue. Since PhoP plays multiple roles in the adaptation of Y. pestis to stressful host conditions, we tested whether this amino acid substitution affects PhoP activity or the ability of Y. pestis to survive in host environments. Compared to the parental KIM6+ strain carrying the modern allele of phoP (phoP-S215), a derivative carrying the basal allele (phoP-G215) exhibited slightly defective growth under a low-Mg2+ condition and decreased transcription of a PhoP target gene, ugd, as well as an ∼8-fold increase in the susceptibility to the antimicrobial peptide polymyxin B. The phoP-G215 strain showed no apparent defect in flea colonization, although a phoP-null mutant showed decreased flea infectivity in competition experiments. Our results suggest that the amino acid variation at position 215 of PhoP causes subtle changes in the PhoP activity and raise the possibility that the change in this residue have contributed to the evolution of increased virulence in Y. pestisIMPORTANCEY. pestis acquired a single nucleotide polymorphism (SNP) in phoP when the highly human-virulent strains diverged from less virulent basal strains, resulting in an amino acid substitution in the DNA binding domain of the PhoP response regulator. We show that Y. pestis carrying the modern phoP allele has an increased ability to induce the PhoP-regulated ugd gene and resist antimicrobial peptides compared to an isogenic strain carrying the basal allele. Given the important roles PhoP plays in host adaptation, the results raise an intriguing possibility that this amino acid substitution contributed to the evolution of increased virulence in Y. pestis Additionally, we present the first evidence that phoP confers a survival fitness advantage to Y. pestis inside the flea midgut.

A high-throughput screening campaign to identify inhibitors of DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD).

The rise of antibacterial resistance among human pathogens represents a problem that could change the landscape of healthcare unless new antibiotics are developed. The methyl erythritol phosphate (MEP) pathway represents an attractive series of targets for novel antibiotic design, considering each enzyme of the pathway is both essential and has no human homologs. Here we describe a pilot scale high-throughput screening (HTS) campaign against the first and second committed steps in the pathway, catalyzed by DXP reductoisomerase (IspC) and MEP cytidylyltransferase (IspD), using compounds present in the commercially available LOPAC1280 library as well as in an in-house natural product extract library. Hit compounds were characterized to deduce their mechanism of inhibition; most function through aggregation. The HTS workflow outlined here is useful for quickly screening a chemical library, while effectively identifying false positive compounds associated with assay constraints and aggregation.

A Fluorescence-Based Assay for Identification of Bacterial Topoisomerase I Poisons.

Bacterial Topoisomerase I is a potential target for the identification of novel topoisomerase poison inhibitors that could provide leads for a new class of antibacterial compounds. Here we describe in detail a fluorescence-based cleavage assay that is successfully used in HTS for the discovery of bacterial topoisomerase Ι poisons.

Plasmid-mediated doxycycline resistance in a Yersinia pestis strain isolated from a rat.

The emergence of antibiotic-resistant Yersinia pestis strains represents a public health concern. Two antibiotic-resistant Y. pestis strains isolated from Madagascar have been previously identified and characterised. Both strains carried conjugative plasmids that conferred resistance to streptomycin or to multiple antibacterial drugs, respectively. Here we characterised a novel Y. pestis strain (IP2180H) that exhibited resistance to doxycycline. This strain was isolated from a rat in Antananarivo (Madagascar) in 1998. Resistance was carried by a conjugative plasmid (pIP2180H) homologous to pB71 from Salmonella enterica. The plasmid of the previously identified streptomycin-resistant Y. pestis strain was also sequenced and it was found that the three antibiotic resistance Y. pestis plasmids sequenced until now are genetically unrelated and are also unrelated to multidrug resistance plasmids from the phylogenetically close bacterial species Yersinia pseudotuberculosis. The fact that the three antibiotic-resistant Malagasy Y. pestis strains were isolated from different hosts, at different times, from distant locations, and carried unrelated plasmids indicates independent horizontal acquisition of genetic material and further demonstrates the capacity of Y. pestis to acquire antibiotic resistance plasmids under natural conditions. Since these resistance plasmids can frequently carry or easily trap antibiotic resistance cassettes, the emergence of new multidrug-resistant Y. pestis strains may be expected and would represent a major health threat.