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Introduction Omidubicel is an advanced cell therapy for allogeneic hematopoietic stem cell transplantation (HSCT), derived from appropriately HLA-matched umbilical cord blood (UCB) and comprised of ex-vivo expanded CD133+ cells and a non-cultured lymphocyte-containing fraction. Recent results of a global phase III trial of omidubicel vs standard UCB showed more rapid hematopoietic recovery, reduced rates of infection, and shorter hospitalizations in patients (pts) randomized to omidubicel (Horwitz et al, Blood, 2021). We now report results of correlative immune reconstitution (IR) studies in this trial (NCT02730299). Methods A total of 125 pts aged 13-65 with hematologic malignancies were randomized to allogeneic transplantation with omidubicel or standard UCB following myeloablative conditioning; 108 pts were transplanted per protocol. An optional IR sub-study was conducted and blood was collected at intervals from Day 7 through one year post-transplant. Cryopreserved samples were analyzed in a central laboratory (Covance) using 16-color and 14-color panels and flow cytometric assays to explore T cell, NK cell, B cell, monocyte, and dendritic cell (DC) subsets. Means, medians, ranges, and standard errors were used to summarize cell counts, and one-tailed t-tests were used to compare counts in the two treatment arms. Results A total of 37 pts from 15 sites consented to the IR sub-study, representing 34% of the per protocol population; 17 pts were transplanted with omidubicel and 20 pts with control (15 [75%] of control with double UCB). Median age was 30 (range: 13-62) years for omidubicel pts and 43 (range: 19-55) years for controls in the sub-study. Median CD3+ content of omidubicel prior to cryopreservation was lower (180 x 10^6; range: 71-580 cells) than that of controls post-thaw (516 x 10^6, range: 183-990 cells). Omidubicel pts achieved neutrophil engraftment at a median of 10 (range: 6-28) days post-transplant compared to a median of 18.5 (range: 14-40) days in controls. Omidubicel pts had fewer BMT-CTN Grade 3 viral infections in the first-year post-transplant than controls (6% vs. 25%, respectively). At Day 7 post-transplant, CD4+ T cell counts were significantly higher in omidubicel pts (37x10^3 cells/ml) than in controls (17x10^3 cells/ml, p=0.011). B cells (12x10^3 vs 1x10^3 cells/ml, p=0.013) and NK cells (6x10^3 vs 3 x10^3 cells/ml, p=0.016), as well as monocyte and DC subsets, were also significantly higher in omidubicel pts (Table). Day 14 results similarly demonstrated higher counts of circulating immune cell subsets in omidubicel pts than in controls (Table). Higher B cell counts were observed in omidubicel pts than in controls at 6 months ([863±463] x10^3 vs. [543±221] x10^3 cells/ml, p=0.03) and one year ([1492±370] x10^3 vs [763±150] x10^3, p=0.02) following transplant (Figure). Conclusions Circulating immune cell subsets were consistently higher in omidubicel pts than controls as early as one week after transplant, and higher B cell counts persisted through one year. These findings correlated with the clinical observation of fewer severe bacterial, fungal, and viral infections in pts treated with omidubicel compared to standard UCB. These results demonstrate that rapid hematopoietic recovery in pts transplanted with omidubicel is accompanied by the early and robust appearance of a broad array of lymphocyte, monocyte, DC, and NK cell subsets, despite substantially fewer numbers of these cells infused, suggesting a facilitator effect of omidubicel on their in vivo expansion. Figure 1 Figure 1. Disclosures Szabolcs: Gamida Cell: Consultancy; Prevail Therapeutics: Consultancy; Sotiria/Forge Biologics: Current equity holder in publicly-traded company. Levy: Gamida Cell: Current Employment. Yackoubov: Gamida Cell: Current Employment. Pato: Gamida Cell: Current Employment. Galamidi-Cohen: Gamida Cell, Ltd: Current Employment. Horwitz: Gamida Cell: Research Funding.

Antibiotics have successfully treated infectious diseases in man, animals and agricultural plants. However, one consequence of usage at any level, subtherapeutic or therapeutic, has been selection of microorganisms resistant to these valuable agents. Today clinicians worldwide face singly resistant and multiply resistant bacteria which complicate treatment of even common infectious agents. This situation calls for a critical evaluation of the numerous ways in which antibiotics are being used so as to evaluate benefits and risks. About half of the antibiotics produced in the United States arc used in animals, chiefly in subtherapeutic amounts for growth promotion. This usage is for prolonged periods leading to selection of multiply-resistant bacteria which enter a common environmental pool. From there, resistance determinants from different sources spread from one bacterium to another, from one animal host to another, from one area to another. The same resistance determinants have been traced to many different genera associated with humans, animals and foods where they pose a continued threat to public health. Since alternative measures for growth promotion, such as antimicrobials which are not used for human therapy and which do not select for multiple-resistances are available, their use, instead of antibiotics, would remove a major factor contributing to the environmental pool of transferable resistance genes.

The division of labour is a central feature of the most sophisticated biological systems, including genomes, multicellular organisms and societies, which took millions of years to evolve. Here we show that a well-organized and robust division of labour can evolve in a matter of days. Mutants emerge within bacterial colonies and work with the parent strain to gain new territory. The two strains self-organize in space: one provides a wetting polymer at the colony edge, whereas the other sits behind and pushes them both along. The emergence of the interaction is repeatable, bidirectional and only requires a single mutation to alter production of the intracellular messenger, cyclic-di-GMP. Our work demonstrates the power of the division of labour to rapidly solve biological problems without the need for long-term evolution or derived sociality. We predict that the division of labour will evolve frequently in microbial populations, where rapid genetic diversification is common., The division of labour—where individuals specialise on different tasks—is fundamental to many sophisticated and ancient biological systems. Here the authors show that bacteria can deploy a robust and functional division of labour in a matter of days via a single mutation.

A series of novel tetracycline derivatives were synthesized with the goal of creating new antibiotics that would be unaffected by the known tetracycline resistance mechanisms. New C-9-position derivatives of minocycline (the aminomethylcyclines [AMCs]) were tested for in vitro activity against Gram-positive strains containing known tetracycline resistance mechanisms of ribosomal protection (Tet M in Staphylococcus aureus , Enterococcus faecalis , and Streptococcus pneumoniae ) and efflux (Tet K in S. aureus and Tet L in E. faecalis ). A number of aminomethylcyclines with potent in vitro activity (MIC range of ≤0.06 to 2.0 μg/ml) were identified. These novel tetracyclines were more active against one or more of the resistant strains than the reference antibiotics tested (MIC range, 16 to 64 μg/ml). The AMC derivatives were active against bacteria resistant to tetracycline by both efflux and ribosomal protection mechanisms. This study identified the AMCs as a novel class of antibiotics evolved from tetracycline that exhibit potent activity in vitro against tetracycline-resistant Gram-positive bacteria, including pathogenic strains of methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE). One derivative, 9-neopentylaminomethylminocycline (generic name omadacycline), was identified and is currently in human trials for acute bacterial skin and skin structure infections (ABSSSI) and community-acquired bacterial pneumonia (CABP).

Natamycin is a poorly soluble, polyene macrolide antifungal agent used in the food industry for the surface treatment of cheese and sausages. This use is not of safety concern. However, highly soluble natamycin–cyclodextrin inclusion complexes have been developed for the protection of beverages. This practice leads to high drug exposures exceeding the safety level. Apart from the definition of an acceptable daily dietary exposure to natamycin, its effect on the faecal flora as a reservoir for resistance has to be examined. Consumption of food to which natamycin has been added and mixed homogeneously, such as yoghurt, and in particular the addition of cyclodextrin inclusion complexes to beverages and wine generates high faecal natamycin concentrations resulting in high drug exposures of faecal Candida spp . Development of natamycin resistance has been observed in Candida spp . colonising the intestinal tract of patients following natamycin treatment of fungal infections. Horizontal gene transfer among different Candida spp. and within Aspergillus fumigatus spreads resistance. Therefore, it cannot be denied that use of natamycin for preservation of yoghurt and beverages may foster development of resistance to polyenes in Candida spp.

Omadacycline is a novel first-in-class aminomethylcycline with potent activity against important skin and pneumonia pathogens, including community-acquired methicillin-resistant Staphylococcus aureus (MRSA), β-hemolytic streptococci, penicillin-resistant Streptococcus pneumoniae , Haemophilus influenzae , and Legionella . In this work, the mechanism of action for omadacycline was further elucidated using a variety of models. Functional assays demonstrated that omadacycline is active against strains expressing the two main forms of tetracycline resistance (efflux and ribosomal protection). Macromolecular synthesis experiments confirmed that the primary effect of omadacycline is on bacterial protein synthesis, inhibiting protein synthesis with a potency greater than that of tetracycline. Biophysical studies with isolated ribosomes confirmed that the binding site for omadacycline is similar to that for tetracycline. In addition, unlike tetracycline, omadacycline is active in vitro in the presence of the ribosomal protection protein Tet(O).

Omadacycline is the first intravenous and oral 9-aminomethylcycline in clinical development for use against multiple infectious diseases including acute bacterial skin and skin structure infections (ABSSSI), community-acquired bacterial pneumonia (CABP), and urinary tract infections (UTI). The comparative in vitro activity of omadacycline was determined against a broad panel of Gram-positive clinical isolates, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Lancefield groups A and B beta-hemolytic streptococci, penicillin-resistant Streptococcus pneumoniae (PRSP), and Haemophilus influenzae ( H. influenzae ). The omadacycline MIC 90 s for MRSA, VRE, and beta-hemolytic streptococci were 1.0 μg/ml, 0.25 μg/ml, and 0.5 μg/ml, respectively, and the omadacycline MIC 90 s for PRSP and H. influenzae were 0.25 μg/ml and 2.0 μg/ml, respectively. Omadacycline was active against organisms demonstrating the two major mechanisms of resistance, ribosomal protection and active tetracycline efflux. In vivo efficacy of omadacycline was demonstrated using an intraperitoneal infection model in mice. A single intravenous dose of omadacycline exhibited efficacy against Streptococcus pneumoniae , Escherichia coli , and Staphylococcus aureus , including tet (M) and tet (K) efflux-containing strains and MRSA strains. The 50% effective doses (ED 50 s) for Streptococcus pneumoniae obtained ranged from 0.45 mg/kg to 3.39 mg/kg, the ED 50 s for Staphylococcus aureus obtained ranged from 0.30 mg/kg to 1.74 mg/kg, and the ED 50 for Escherichia coli was 2.02 mg/kg. These results demonstrate potent in vivo efficacy including activity against strains containing common resistance determinants. Omadacycline demonstrated in vitro activity against a broad range of Gram-positive and select Gram-negative pathogens, including resistance determinant-containing strains, and this activity translated to potent efficacy in vivo .

Multidrug efflux pumps mediate resistance to antibiotics and other toxic compounds. We studied the role of AcrAB-TolC, the main efflux pump in Escherichia coli, in regulating gene expression.Deletion mutants, an acrABp-lacZ fusion and reverse transcription-real-time quantitative PCR experiments were used to study the role of AcrAB-TolC and metabolism in regulating gene expression of the acrAB operon and its transcriptional regulators.Deletion of the acrB gene increased the expression of the acrAB operon. A similar induction of acrAB was found when acrA or tolC was deleted, and when the pump function was inhibited using phenylalanine-arginine-β-naphthylamide. The induction of acrAB in the ΔacrB strain was totally (AcrR or SoxS) or partially (SoxR or MarA) prevented when the genes for these acrAB regulators were also deleted. The expression of soxS and marA, but not of acrR, was increased in the ΔacrB strain, which also showed altered expression of many other genes related to different cellular processes, including motility. Deletion of the metabolic genes entA and entE (enterobactin biosysnthesis), glpX (gluconeogenesis), cysH (cysteine biosynthesis) and purA (purine biosynthesis) also prevented activation of the acrAB promoter in the ΔacrB strain. Addition of the enterobactin biosynthesis intermediate metabolite 2,3-dihydroxybenzoate induced the expression of acrAB.These results together suggest a model in which the AcrAB-TolC pump effluxes cellular metabolites that are toxic and/or have a signalling role. If the pump is inactivated or inhibited, these metabolites would accumulate, inactivating AcrR and/or up-regulating soxS and marA expression, ultimately triggering the up-regulation of acrAB expression to restore homeostasis.

The Escherichia coli regulator MarR represses the multiple-antibiotic resistance operon marRAB and responds to phenolic compounds, including sodium salicylate, which inhibit its activity. Crystals obtained in the presence of a high concentration of salicylate indicated two possible salicylate sites, SAL-A and SAL-B. However, it was unclear whether these sites were physiologically significant or were simply a result of the crystallization conditions. A study carried out on MarR homologue MTH313 suggested the presence of a salicylate binding site buried at the interface between the dimerization and the DNA-binding domains. Interestingly, the authors of the study indicated a similar pocket conserved in the MarR structure. Since no mutagenesis analysis had been performed to test which amino acids were essential in salicylate binding, we examined the role of residues that could potentially interact with salicylate. We demonstrated that mutations in residues shown as interacting with salicylate at SAL-A and SAL-B in the MarR-salicylate structure had no effect on salicylate binding, indicating that these sites were not the physiological regulatory sites. However, some of these residues (P57, R86, M74, and R77) were important for DNA binding. Furthermore, mutations in residues R16, D26, and K44 significantly reduced binding to both salicylate and 2,4-dinitrophenol, while a mutation in residue H19 impaired the binding to 2,4-dinitrophenol only. These findings indicate, as for MTH313, the presence of a ligand binding pocket located between the dimerization and DNA binding domains.

The marRAB operon is conserved in seven genera of enteric bacteria (Escherichia, Shigella, Klebsiella, Enterobacter, Salmonella, Cronobacter, and Citrobacter). MarA is a transcriptional regulator affecting many genes involved in resistance to stresses, and MarR is an autorepressor of the operon, but a role for the marB gene has been unclear. A recent work reported that deletion of marB causes resistance to certain stresses and increases the amount of marA transcript. We show here that the small (216 bp) marB gene encodes a protein, not an sRNA, because two different stop codons within the predicted open reading frame of marB prevented plasmid-borne marB from complementing ΔmarB::Kan. The ΔmarB::Kan mutation did not increase the stability of the marA transcript, suggesting that MarB does not destabilize the marA transcript but rather reduces its rate of transcription. Placing the putative signal sequence of MarB upstream of signal-sequence-less alkaline phosphatase guided the phosphatase to its normal periplasmic location. We conclude that MarB is a small periplasmic protein that represses the marRAB promoter by an indirect mechanism, possibly involving a signal to one of the cytoplasmic regulators of that promoter.

The Food and Drug Administration (FDA) recently released a final rule to ban triclosan and 18 other antimicrobial chemicals from soaps. We applaud this rule specifically because of the associated risks that triclosan poses to the spread of antibiotic resistance throughout the environment. This persistent chemical constantly stresses bacteria to adapt, and behavior that promotes antibiotic resistance needs to be stopped immediately when the benefits are null.

Persister cells are highly tolerant to different antibiotics and are associated with relapsing infections. In order to understand this phenomenon further, we exposed a transposon library to a lethal concentration of ampicillin, and mutants that survived were identified by transposon sequencing (Tn-Seq). We determined that mutations related to carbon metabolism, cell envelope (cell wall generation and membrane proteins), and stress response have a role in persister cell generation.

Comparison of the genome sequences of three Pseudomonas fluorescens strains reveals a heterogeneity reminiscent of a species complex rather than a single species, Background Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species. Results Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome. Conclusions P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.

Pseudomonas spp. adapt rapidly to environmental fluctuations. Loss or overproduction of polyphosphate reduces the fitness of Pseudomonas fluorescens Pf0-1, indicating the importance of the fine-tuning of polyphosphate production. An antisense RNA was investigated and shown to regulate the polyphosphate kinase gene ( ppk ) by a posttranscriptional mechanism reducing ppk transcript abundance.

AdnA in Pseudomonas fluorescens , an ortholog of FleQ in P. aeruginosa , regulates both motility and flagellum-mediated attachment to various surfaces. A whole-genome microarray determined the AdnA transcriptome by comparing the gene expression pattern of wild-type Pf0-1 to that of Pf0-2x ( adnA deletion mutant) in broth culture. In the absence of AdnA, expression of 92 genes was decreased, while 11 genes showed increased expression. Analysis of 16 of these genes fused to lacZ confirmed the microarray results. Several genes were further evaluated for their role in motility and biofilm formation. Two genes, Pfl01_1508 and Pfl01_1517, affected motility and had different effects on biofilm formation in Pf0-1. These two genes are predicted to specify proteins similar to the glycosyl transferases FgtA1 and FgtA2, which have been shown to be involved in virulence and motility in P. syringae . Three other genes, Pfl01_1516, Pfl01_1572, and Pfl01_1573, not previously associated with motility and biofilm formation in Pseudomonas had similar effects on biofilm formation in Pf0-1. Deletion of each of these genes led to different motility defects. Our data revealed an additional level of complexity in the control of flagellum function beyond the core genes known to be required and may yield insights into processes important for environmental persistence of P. fluorescens Pf0-1.

Paralogous transcriptional regulators MarA, Rob, and SoxS act individually and together to control expression of more than 80 Escherichia coli genes. Deletion of marA, rob, and soxS from an E. coli clinical isolate prevents persistence beyond 2 days postinfection in a mouse model of pyelonephritis. We used microarray analysis to identify 242 genes differentially expressed between the triple deletion mutant and its parent strain at 2 days postinfection in the kidney. One of these, znuC of the zinc transport system ZnuACB, displayed decreased expression in the triple mutant compared to that in the parental strain, and deletion of znuC from the parental strain reduced persistence. The marA rob soxS triple deletion mutant was less viable in vitro under limited-Zn and Zn-depleted conditions, while disruption of znuC caused a reduction in the growth rates for the parental and triple mutant strains to equally low levels under limited-Zn or Zn-depleted conditions. Complementation of the triple mutant with soxS, but not marA or rob, restored the parental growth rate in Zn-depleted medium, while deletion of only soxS from the parental strain led to low growth in Zn-depleted medium. Both results suggested that SoxS is a major regulator responsible for growth under Zn-depleted conditions. Gel shift experiments failed to show direct binding of SoxS to the znuCB promoter, thus suggesting indirect control of znuCB expression by SoxS. While SoxS expression in the triple mutant fully restored persistence, increased expression of znuACB via a plasmid in this mutant only partially restored wild-type levels of persistence in the kidney. This work implicates SoxS control of znuCB expression as a key factor in persistence of E. coli in murine pyelonephritis.

The history of the tetracyclines involves the collective contributions of thousands of dedicated researchers, scientists, clinicians, and business executives over the course of more than 60 years. Discovered as natural products from actinomycetes soil bacteria, the tetracyclines were first reported in the scientific literature in 1948. They were noted for their broad spectrum antibacterial activity and were commercialized with clinical success beginning in the late 1940s to the early 1950s. The second-generation semisynthetic analogs and more recent third-generation compounds show the continued evolution of the tetracycline scaffold toward derivatives with increased potency as well as efficacy against tetracycline-resistant bacteria, with improved pharmacokinetic and chemical properties. Their biologic activity against a wide spectrum of microbial pathogens and their uses in mammalian models of inflammation, neurodegeneration, and other biological systems indicate that the tetracyclines will continue to be successful therapeutics in infectious diseases and as potential therapeutics against inflammation-based mammalian cell diseases.

The original and successful business model of return on investment being sufficiently attractive to the pharmaceutical industry to encourage development of new antibacterial molecules and related diagnostics has been compromised by increasing development costs and regulatory hurdles, resulting in a decreasing chance of success and financial return. The supply of new effective agents is diminishing along with the number of companies engaged in antibacterial research and development. The BSAC Working Party on The Urgent Need:Regenerating Antibacterial Drug Discovery and Development identified the need to establish, communicate and apply the true health and economic value of antibacterials, along with the adoption of meaningful incentives, as part of the future model for antibacterial development. Robust data are needed on the cost of resistance and ineffective treatment of bacterial infection, along with national and local holistic analyses of the cost-benefit of antibacterials. An understanding of the true health and economic value of antibacterials and the cost of resistance across healthcare systems needs to be generated, communicated and used in order to set a pricing and reimbursement structure that is commensurate with value. The development and economic model of antibacterial use needs to be rebuilt based on this value through dialogue with the various stakeholders, including the pharmaceutical industry, and alternative incentives from 'push' to 'pull' and funding models, such as public/private partnerships, agreed. A research and development model that succeeds in developing and delivering new antibacterial agents that address the health needs of society from start to finish, 'from cradle to grave', must be established.

The ability of soil bacteria to successfully compete with a range of other microbial species is crucial for their growth and survival in the nutrient-limited soil environment. In the present work, we studied the behavior and transcriptional responses of soil-inhabiting Pseudomonas fluorescens strain Pf0-1 on nutrient-poor agar to confrontation with strains of three phylogenetically different bacterial genera, that is, Bacillus, Brevundimonas and Pedobacter. Competition for nutrients was apparent as all three bacterial genera had a negative effect on the density of P. fluorescens Pf0-1; this effect was most strong during the interaction with Bacillus. Microarray-based analyses indicated strong differences in the transcriptional responses of Pf0-1 to the different competitors. There was higher similarity in the gene expression response of P. fluorescens Pf0-1 to the Gram-negative bacteria as compared with the Gram-positive strain. The Gram-negative strains did also trigger the production of an unknown broad-spectrum antibiotic in Pf0-1. More detailed analysis indicated that expression of specific Pf0-1 genes involved in signal transduction and secondary metabolite production was strongly affected by the competitors' identity, suggesting that Pf0-1 can distinguish among different competitors and fine-tune its competitive strategies. The results presented here demonstrate that P. fluorescens Pf0-1 shows a species-specific transcriptional and metabolic response to bacterial competitors and provide new leads in the identification of specific cues in bacteria-bacteria interactions and of novel competitive strategies, antimicrobial traits and genes.

The MarA protein of Escherichia coli can both activate and repress the initiation of transcription, depending on the position and orientation of its degenerate 20-bp binding site (“marbox”) at the promoter. For all three known repressed genes, the marbox overlaps the promoter. It has been reported that MarA represses the rob promoter via an RNA polymerase (RNAP)-DNA-MarA ternary complex. Under similar conditions, we found a ternary complex for the repressed purA promoter also. These findings, together with the backwards orientation of repressed marboxes, suggested a unique interaction of MarA with RNAP in repression. However, no repression-specific residues of MarA could be found among 38 single-alanine replacement mutations previously shown to retain activation function or among mutants from random mutagenesis. Mutations Thr12Ala, Arg36Ala, Thr95Ile, and Pro106Ala were more damaging for activation than for repression, some up to 10-fold, so these residues may play a specific role in activation. We found that nonspecific binding of RNAP to promoterless regions of DNA was presumably responsible for the ternary complexes seen previously. When RNAP binding was promoter specific, MarA reduced RNAP access to the rob promoter; there was little or no ternary complex. These findings strongly implicate steric hindrance as the mechanism of repression of rob by MarA.

ExsA is a multiple adaptational response (MAR) transcription factor, regulating the expression of a virulence determinant, the type III secretion system (T3SS) in Pseudomonas aeruginosa. Non-cytotoxic, non-antibacterial N-hydroxybenzimidazoles were identified as effective inhibitors of ExsA-DNA binding, and their potential utility as anti-virulence agents for P. aeruginosa was demonstrated in a whole cell assay. Select N-hydroxybenzimidazole inhibitors were stable in an in vitro human liver microsomal assay.

Multidrug resistance (MDR) in clinical isolates of Escherichia coli can be associated with overexpression of marA , a transcription factor that upregulates multidrug efflux and downregulates membrane permeability. Using random transposome mutagenesis, we found that many chromosomal genes and environmental stimuli affected MarA-mediated antibiotic resistance. Seven genes affected resistance mediated by MarA in an antibiotic-specific way; these were mostly genes encoding unrelated enzymes, transporters, and unknown proteins. Other genes affected MarA-mediated resistance to all antibiotics tested. These genes were acrA , acrB , and tolC (which encode the major MarA-regulated multidrug efflux pump AcrAB-TolC), crp , cyaA , hns , and pcnB (four genes involved in global regulation of gene expression), and the unknown gene damX . The last five genes affected MarA-mediated MDR by altering marA expression or MarA function specifically on acrA . These findings demonstrate that MarA-mediated MDR is regulated at multiple levels by different genes and stimuli, which makes it both complex and fine-tuned and interconnects it with global cell regulation and metabolism. Such a regulation could contribute to the adaptation and spread of MDR strains and may be targeted to treat antibiotic-resistant E. coli and related pathogens.

Cationic antimicrobial peptides (CAMPs), a component of the mammalian immune system, protect the host from bacterial infections. The roles of the Escherichia coli transcriptional regulators MarA, SoxS and Rob in susceptibility to these peptides were examined. Overexpression of marA, either in an antibiotic-resistant marR mutant or from a plasmid, decreased bacterial susceptibility to CAMPs. Overexpression of the soxS gene from a plasmid, which decreased susceptibility to antibiotics, unexpectedly caused no decrease in CAMP susceptibility; instead it produced increased susceptibility to different CAMPs. Deletion or overexpression of rob had little effect on CAMP susceptibility. The marRAB operon was upregulated when E. coli was incubated in sublethal amounts of CAMPs polymyxin B, LL-37 or human β-defensin-1; however, this upregulation required Rob. Deletion of acrAB increased bacterial susceptibility to polymyxin B, LL-37 and human β-defensin-1 peptides. Deletion of tolC yielded an even greater increase in susceptibility to these peptides and also led to increased susceptibility to human α-defensin-2. Inhibition of cellular proton-motive force increased peptide susceptibility for wild-type and acrAB deletion strains; however, it decreased susceptibility of tolC mutants. These findings demonstrate that CAMPs are both inducers of marA-mediated drug resistance through interaction with Rob and also substrates for efflux in E. coli. The three related transcriptional regulators show different effects on bacterial cell susceptibility to CAMPs.

Transposon inactivation of ycgE , a gene encoding a putative transcriptional regulator, led to decreased multidrug susceptibility in an Escherichia coli lon mutant. The multidrug susceptibility phenotype (e.g., to tetracycline and β-lactam antibiotics) required the inactivation of both lon and ycgE . In this mutant, a decreased amount of OmpF porin contributes to the lowered drug susceptibility, with a greater effect at 26°C than at 37°C.

Background Organisms resistant to antimicrobials continue to emerge and spread. This study was performed to measure the medical and societal cost attributable to antimicrobial-resistant infection (ARI). Methods A sample of high-risk hospitalized adult patients was selected. Measurements included ARI, total cost, duration of stay, comorbidities, acute pathophysiology, Acute Physiology and Chronic Health Evaluation III score, intensive care unit stay, surgery, health care-acquired infection, and mortality. Hospital services used and outcomes were abstracted from electronic and written medical records. Medical costs were measured from the hospital perspective. A sensitivity analysis including 3 study designs was conducted. Regression was used to adjust for potential confounding in the random sample and in the sample expanded with additional patients with ARI. Propensity scores were used to select matched control subjects for each patient with ARI for a comparison of mean cost for patients with and without ARI. Results In a sample of 1391 patients, 188 (13.5%) had ARI. The medical costs attributable to ARI ranged from $18,588 to $29,069 per patient in the sensitivity analysis. Excess duration of hospital stay was 6.4-12.7 days, and attributable mortality was 6.5%. The societal costs were $10.7-$15.0 million. Using the lowest estimates from the sensitivity analysis resulted in a total cost of $13.35 million in 2008 dollars in this patient cohort. Conclusions The attributable medical and societal costs of ARI are considerable. Data from this analysis could form the basis for a more comprehensive evaluation of the cost of resistance and the potential economic benefits of prevention programs.

LcrF, a Multiple Adaptational Response (MAR) transcription factor, regulates virulence in Yersinia pestis and Yersinia pseudotuberculosis. In a search for small molecule inhibitors of LcrF, an acrylic amide series of N-hydroxybenzimidazoles was synthesized, and the SAR (structure-activity relationship) was examined. Selected test compounds demonstrated inhibitory activity in a primary cell-free LcrF-DNA binding assay as well as in a secondary whole cell assay (Type III secretion system dependent Y. pseudotuberculosis cytotoxicity assay). The inhibitors exhibited no measurable antibacterial activity in vitro, confirming that they do not target bacterial growth. These results demonstrate that N-hydroxybenzimidazole inhibitors, exemplified by 14, 22 and 36, are effective anti-virulence agents, and have the potential to prevent infections caused by Yersinia spp.

Knowledge of the genetic basis for bacterial survival and persistence in soil is a critical component in the development of successful biological control strategies and for understanding the ecological success of bacteria. We found a locus specifying polyphosphate kinase ( ppk ) and a nonpredicted antisense RNA ( iiv8 ) in Pseudomonas fluorescens Pf0-1 to be necessary for optimal competitive fitness in LB broth culture and sterile loam soil. Pf0-1 lacking ppk and iiv8 was more than 10-fold less competitive against wild-type Pf0-1 in sterile loam soil low in inorganic phosphate. Studies indicated that ppk , and not iiv8 , was required for competitive fitness. No role for iiv8 was identified. While a ppk and iiv8 mutant of Pf0-1 did not have increased sensitivity to osmotic, oxidative, and acid stress, it was more sensitive to elevated temperatures in laboratory medium and during growth in sterile soil. ppk was shown to be part of the Pho regulon in P. fluorescens , being upregulated in response to a low external P i concentration. Of importance, overproduction of polyphosphate in the soil environment appears to be more deleterious than production of none at all. Our findings reveal a new role for polyphosphate (and the need for proper regulation of its production) in competitive fitness of P. fluorescens in laboratory and soil environments.

Antibiotic resistance, reported for sulfonamides in the mid-1930s and for penicillins in the 1940s, remains a stubborn quandary. What was once confined mainly to hospitals increasingly involves multidrug resistance that encompasses communities and encircles the globe. Virtually all types of bacterial infections are becoming resistant to antibiotic treatments, according to officials at the Centers for Disease Control and Prevention in Atlanta, Ga. Yet, despite decades of grappling with these issues, we still do not understand fully how genes carrying resistance traits spread, what makes certain species highly promiscuous in transferring those traits, whether there are effective barriers to their spread, and the frequency with which resistance genes move independently or in tandem with other migrating genes.

Healthcare-associated infections (HAIs) have been a hot topic for several decades. An understanding of HAIs should be based on an understanding of the organisms that cause infection and determine prevention. Although some improvements in control in hospitals have been recorded, the community setting is now implicated, and the role of microbiology in diagnosis, detection of carriers and strain typing of organisms is evident. As healthcare systems vary widely, prevention strategies must be designed accordingly. Hand hygiene, however, remains applicable in all settings, and the WHO is strongly promoting alcohol-based hand rubs to interrupt transmission. Some countries are only beginning to develop standards, whereas compliance is obligatory in others. Economics and cost factors are common to all countries, and litigation is increasingly a factor in some.

The annotation process of a newly sequenced bacterial genome is largely based on algorithms derived from databases of previously defined RNA and protein-encoding gene structures. This process generally excludes the possibility that the two strands of a given stretch of DNA can each harbor a gene in an overlapping manner. While the presence of such structures in eukaryotic genomes is considered to be relatively common, their counterparts in prokaryotic genomes are just beginning to be recognized. Application of an in vivo expression technology has previously identified 22 discrete genetic loci in Pseudomonas fluorescens Pf0-1 that were specifically activated in the soil environment, of which 10 were present in an antisense orientation relative to previously annotated genes. This observation led to the hypothesis that the physiological role of overlapping genetic structures may be relevant to growth conditions outside artificial laboratory media. Here, we examined the role of one of the overlapping gene pairs, iiv19 and leuA2 , in soil. Although iiv19 was previously demonstrated to be preferentially activated in the soil environment, its absence did not alter the ability of P. fluorescens to colonize or survive in soil. Surprisingly, the absence of the leuA2 gene conferred a fitness advantage in the soil environment when leucine was supplied exogenously. This effect was determined to be independent of the iiv19 gene, and further analyses revealed that amino acid antagonism was the underlying mechanism behind the observed fitness advantage of the bacterium in soil. Our findings provide a potential mechanism for the frequent occurrence of auxotrophic mutants of Pseudomonas spp. in the lungs of cystic fibrosis patients.

MarA, a transcriptional regulator in Escherichia coli , affects functions such as multiple-antibiotic resistance (Mar) and virulence. Usually an activator, MarA is a repressor of hdeAB and other acid resistance genes. We found that, in wild-type cells grown in LB medium at pH 7.0 or pH 5.5, repression of hdeAB by MarA occurred only in stationary phase and was reduced in the absence of H-NS and GadE, the main regulators of hdeAB . Moreover, repression of hdeAB by MarA was greater in the absence of GadX or Lrp in exponential phase at pH 7.0 and in the absence of GadW or RpoS in stationary phase at pH 5.5. In turn, MarA enhanced repression of hdeAB by H-NS and hindered activation by GadE in stationary phase and also reduced the activity of GadX, GadW, RpoS, and Lrp on hdeAB under some conditions. As a result of its direct and indirect effects, overexpression of MarA prevented most of the induction of hdeAB expression as cells entered stationary phase and made the cells sevenfold more sensitive to acid challenge at pH 2.5. These findings show that repression of hdeAB by MarA depends on pH, growth phase, and other regulators of hdeAB and is associated with reduced resistance to acid conditions.

Structure-based drug design was utilized to identify potent small-molecule inhibitors of proteins within the AraC family of bacterial transcription factors, which control virulence in medically important microbes. These agents represent a novel approach to fight infectious disease and may be less likely to promote resistance development. These compounds lack intrinsic antibacterial activity in vitro and were able to limit a bacterial infection in a mouse model of urinary tract infection.

The Escherichia coli marRAB operon specifies two regulatory proteins, MarR (which represses) and MarA (which activates expression of the operon). The latter controls expression of multiple other chromosomal genes implicated in cell physiology, multiple drug resistance and virulence. Using randomly cloned E. coli DNA fragments in the bacterial adenylate cyclase two-hybrid system, we found that transketolase A (TktA) interacts with MarR. Purified (6H)-TktA immobilized on NiNTA resin-bound MarR. Overexpression or deletion of tktA showed that TktA interfered with MarR repression of the marRAB operon. Deletion of tktA increased antibiotic and oxidative stress susceptibilities, while its overexpression decreased them. Hydrogen peroxide induced tktA at 1 h treatment, while an increase in marRAB expression occurred only after 3 h exposure. This increase was dependent on the presence of tktA. Two MarR mutations which eliminated MarR binding to the marRAB operator and one which decreased dimerization of MarR had no effect on MarR interaction with TktA in the two-hybrid system. However, the interaction was disrupted by one of the three tested superrepressor mutant MarR proteins known to increase MarR binding to DNA. TktA inhibition of repression by MarR demonstrates a previously unrecognized level of control of the expression of marRAB operon.

Background. Much has been written recently about the potential hazards versus benefits of antibacterial (biocide)‐containing soaps. The purpose of this systematic literature review was to assess the studies that have examined the efficacy of products containing triclosan, compared with that of plain soap, in the community setting, as well as to evaluate findings that address potential hazards of this use—namely, the emergence of antibioticresistant bacteria. Methods. The PubMed database was searched for English-language articles, using relevant keyword combinations for articles published between 1980 and 2006. Twenty-seven studies were eventually identified as being relevant to the review. Results. Soaps containing triclosan within the range of concentrations commonly used in the community setting (0.1%‐0.45% wt/vol) were no more effective than plain soap at preventing infectious illness symptoms and reducing bacterial levels on the hands. Several laboratory studies demonstrated evidence of triclosan-adapted crossresistance to antibiotics among different species of bacteria. Conclusions. The lack of an additional health benefit associated with the use of triclosan-containing consumer soaps over regular soap, coupled with laboratory data demonstrating a potential risk of selecting for drug resistance, warrants further evaluation by governmental regulators regarding antibacterial product claims and advertising. Further studies of this issue are encouraged.

MarA, SoxS and Rob are transcription factors belonging to the AraC family. While these proteins have been associated historically with control of multiple antibiotic resistance, and tolerance to oxidative stress agents and organic solvents, only a paucity of experimental data support a role in regulating virulence. Clinical Escherichia coli isolates, and isogenic strains lacking marA, soxS and rob, were studied in a murine model of ascending pyelonephritis, which is a clinically relevant model of urinary tract infection. Organisms lacking all three transcription factors (triple knockouts) were significantly less virulent than parental strains, and complementation studies demonstrated that the addition of marA, soxS and rob individually restored wild-type virulence in the triple-knockout strain. Deletion of soxS or rob alone was more detrimental than the removal of marA. Thus, all three proteins contribute to virulence in vivo.

A spontaneous mutant (M113) of Escherichia coli AG100 with an unstable multiple antibiotic resistance (Mar) phenotype was isolated in the presence of tetracycline. Two mutations were found: an insertion in the promoter of lon ( lon3 ::IS 186 ) that occurred first and a subsequent large tandem duplication, dupIS 186 , bearing the genes acrAB and extending from the lon3 ::IS 186 to another IS 186 present 149 kb away from lon . The decreased amount of Lon protease increased the amount of MarA by stabilization of the basal quantities of MarA produced, which in turn increased the amount of multidrug effux pump AcrAB-TolC. However, in a mutant carrying only a lon mutation, the overproduced pump mediated little, if any, increased multidrug resistance, indicating that the Lon protease was required for the function of the pump. This requirement was only partial since resistance was mediated when amounts of AcrAB in a lon mutant were further increased by a second mutation. In M113, amplification of acrAB on the duplication led to increased amounts of AcrAB and multidrug resistance. Spontaneous gene duplication represents a new mechanism for mediating multidrug resistance in E. coli through AcrAB-TolC.

A battle to control and curtail bacterial infectious diseases is being waged in our hospitals and communities through antibiotic therapies and vaccines targeting specific species. But what effects do these interventions have on the epidemiology of infections caused by the organisms that are part of our natural microbial flora? Gram-positive and gram-negative bacteria appear as new disease agents from among commensal flora. These include vancomycin resistant enterococci (VRE), community-associated methicillin resistant Staphylococcus aureus (CA-MRSA), non-vaccine invasive serotypes of Streptococcus pneumoniae, new strains of non-type b Haemophilus influenzae and multi-drug resistant Escherichia coli. These examples illustrate how clinical improvements and widespread use and misuse of antibiotics have pushed evolution, allowing normally non-pathogenic strains to become infectious disease threats to human health.

Targeting components of the infectious process as a means to prevent infection has long been considered as an alternative to classic antimicrobial therapies. Although no clinically used drugs have yet emerged from these efforts, the dwindling supply of anti-infective treatment options within the physician's armamentarium has stoked a renewed interest in the identification and development of novel strategies to prevent infection.

Studies were undertaken to determine the genetic needs for the survival of Pseudomonas fluorescens Pf0-1, a gram-negative soil bacterium potentially important for biocontrol and bioremediation, in soil. In vivo expression technology (IVET) identified 22 genes with elevated expression in soil relative to laboratory media. Soil-induced sequences included genes with probable functions of nutrient acquisition and use, and of gene regulation. Ten sequences, lacking similarity to known genes, overlapped divergent known genes, revealing a novel genetic organization at those soil-induced loci. Mutations in three soil-induced genes led to impaired early growth in soil but had no impact on growth in laboratory media. Thus, IVET studies have identified sequences important for soil growth and have revealed a gene organization that was undetected by traditional laboratory approaches.