Andrea Marra

A genomic analysis of two‐component signal transduction in Streptococcus pneumoniae

A genomics‐based approach was used to identify the entire gene complement of putative two‐component signal transduction systems (TCSTSs) in Streptococcus pneumoniae. A total of 14 open reading frames (ORFs) were identified as putative response regulators, 13 of which were adjacent to genes encoding probable histidine kinases. Both the histidine kinase and response regulator proteins were categorized into subfamilies on the basis of phylogeny. Through a systematic programme of mutagenesis, the importance of each novel TCSTS was determined with respect to viability and pathogenicity. One TCSTS was identified that was essential for the growth of S. pneumoniaeThis locus was highly homologous to the yycFG gene pair encoding the essential response regulator/histidine kinase proteins identified in Bacillus subtilis and Staphylococcus aureus. Separate deletions of eight other loci led in each case to a dramatic attenuation of growth in a mouse respiratory tract infection model, suggesting that these signal transduction systems are important for the in vivo adaptation and pathogenesis of S. pneumoniae. The identification of conserved TCSTSs important for both pathogenicity and viability in a Gram‐positive pathogen highlights the potential of two‐component signal transduction as a multicomponent target for antibacterial drug discovery.

Acinetobacter baumannii: an emerging multidrug-resistant threat

Amid the recent attention focused on the growing impact of methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa infections, the pathogen Acinetobacter baumannii has been stealthily gaining ground as an agent of serious nosocomial and community-acquired infection. Historically, Acinetobacter spp. have been associated with opportunistic infections that were rare and of modest severity; the last two decades have seen an increase in both the incidence and seriousness of A. baumannii infection, with the main targets being patients in intensive-care units. Although this organism appears to have a predilection for the most vulnerable patients, community-acquired A. baumannii infection is an increasing cause for concern. The increase in A. baumannii infections has paralleled the alarming development of resistance it has demonstrated. The persistence of this organism in healthcare facilities, its inherent hardiness and its resistance to antibiotics results in it being a formidable emerging pathogen. This review aims to put into perspective the threat posed by this organism in hospital and community settings, describes new information that is changing our view of Acinetobacter virulence and resistance, and calls for greater understanding of how this multifaceted organism came to be a major pathogen.

Differential fluorescence induction reveals Streptococcus pneumoniae loci regulated by competence stimulatory peptide

Differential fluorescence induction (DFI) in Streptococcus pneumoniae was used as a method for the discovery of genes activated in specific growth environments. Competence stimulatory peptide (CSP) was used as the model inducing system to identify differentially expressed genes. To identify CSP‐induced promoters, a plasmid library was constructed by inserting random pieces of S. pneumoniae chromosomal DNA upstream of the promoterless gfpmut2 gene in an Escherichia coli/S. pneumoniae shuttle vector. S. pneumoniae carrying the library were induced with CSP and enriched for green fluorescent protein (GFP)‐expressing bacteria using fluorescence‐activated cell sorting. A total of 886 fluorescent clones was screened, and 12 differentially activated promoter elements were identified. Sequence analysis of these clones revealed that three were associated with novel competence loci, one of which we show is essential for DNA uptake, and six are known CSP‐inducible promoters. We also explored whether competence proteins have a role in virulence and found that mutations in three CSP‐inducible genes resulted in attenuated virulence phenotypes in either of two murine infection models. These results demonstrate the utility of DFI as a method for identifying differentially expressed genes in S. pneumoniae and the potential utility of applying DFI to other Gram‐positive bacteria.

Potent Inhibitors of LpxC for the Treatment of Gram-Negative Infections

In this paper, we present the synthesis and SAR as well as selectivity, pharmacokinetic, and infection model data for representative analogues of a novel series of potent antibacterial LpxC inhibitors represented by hydroxamic acid.

Discovery of azetidinyl ketolides for the treatment of susceptible and multidrug resistant community-acquired respiratory tract infections.

Respiratory tract bacterial strains are becoming increasingly resistant to currently marketed macrolide antibiotics. The current alternative telithromycin (1) from the newer ketolide class of macrolides addresses resistance but is hampered by serious safety concerns, hepatotoxicity in particular. We have discovered a novel series of azetidinyl ketolides that focus on mitigation of hepatotoxicity by minimizing hepatic turnover and time-dependent inactivation of CYP3A isoforms in the liver without compromising the potency and efficacy of 1.

Targeting Virulence for Antibacterial Chemotherapy

The antibacterial drug discovery industry is fast losing participants; at the same time it is facing the challenge of developing new antibiotics that are effective against frequently occurring and multiply resistant organisms. One intriguing approach is to target bacterial virulence, and the last decade or so has seen a focus on bacterial pathogenesis along with the development of reagents and strategies that could make this possible. Several processes utilised by a range of bacteria to cause infection may be conserved enough to make attractive targets; indeed it is known that mammalian cells can affect bacterial gene expression and vice versa. Interesting targets involving virulence include type III secretion systems, two-component signal transduction systems, quorum sensing, and biofilm formation. In order to better understand these systems and strategies, investigators have developed novel strategies of their own, involving negative selections, surrogate models of infection, and screens for gene induction and antigenicity. Inhibitors of such targets would be unlikely to adversely affect patients, be cross-resistant to existing therapies, or cause resistance themselves. It might be the case that virulence target-based therapies would not be powerful enough to clear an existing infection alone, but if they are instead considered as adjunct therapy to existing antibiotics, or potentiators of the host immune response, they may show efficacy in a non-traditional way.

Preparation, Gram-Negative Antibacterial Activity, and Hydrolytic Stability of Novel Siderophore-Conjugated Monocarbam Diols

A novel series of monocarbam compounds exhibiting promising antibacterial activity against multidrug resistant Gram-negative microorganisms is reported, along with the synthesis of one such molecule MC-1 (1). Also reported are structure-activity relationships associated with the in vitro and in vivo efficacy of 1 and related analogues in addition to the hydrolytic stability of such compounds and possible implications thereof.

Can virulence factors be viable antibacterial targets

There is a real crisis in healthcare with the emergence of bacterial pathogens resistant to multiple drugs. The drug discovery industry is faced with the challenge of developing new classes of antibiotics that are effective against resistant organisms. Targeting bacterial virulence is one approach that has yet to be fully exploited, and the last decade or so has seen the development of reagents, screens and approaches that could make this possible. Several processes utilized by bacteria to cause infection are employed in a wide range of pathogens and as such may make attractive targets. Inhibitors of such targets would be unlikely to affect host cells, be cross-resistant to existing therapies and induce resistance themselves.

Effect of Linezolid on the 50% Lethal Dose and 50% Protective Dose in Treatment of Infections by Gram-Negative Pathogens in Naive and Immunosuppressed Mice and on the Efficacy of Ciprofloxacin in an Acute Murine Model of Septicemia

ABSTRACT Murine models of infection were used to study the effect of linezolid on the virulence of Gram-negative bacteria and to assess potential pharmacodynamic interactions with ciprofloxacin in the treatment of these infections, prompted by observations from a recent clinical trial. Naive and immunosuppressed mice were challenged with Klebsiella pneumoniae 53A1109, K. pneumoniae GC6658, and Pseudomonas aeruginosa UC12120 in acute sepsis and pulmonary infection models, using different serial dilutions of these pathogens (groups of 8 animals each). Linezolid (100 mg/kg/dose) was administered orally at 0.5 and 4.0 h postchallenge in the sepsis model and at 4 h postchallenge followed by 2 days of twice-daily treatment in the pulmonary model. Further, ciprofloxacin alone and in combination with oral linezolid was investigated in the sepsis model. Survival was assessed for 4 and 10 days postchallenge in the systemic and respiratory models, respectively. The data were fitted to a nonlinear regression analysis to determine 50% lethal doses (LD50s) and 50% protective doses (PD50s). A clinically relevant, high-dose regimen of linezolid had no significant effect on LD50 in these models. This lack of effect was independent of immune status. A combination of oral ciprofloxacin with linezolid yielded lower PD50s than oral ciprofloxacin alone (ciprofloxacin in combination, 8.4 to 32.7 mg/kg; oral ciprofloxacin, 39.4 to 88.3 mg/kg). Linezolid did not improve the efficacy of subcutaneous ciprofloxacin (ciprofloxacin in combination, 2.0 to 2.4 mg/kg; subcutaneous ciprofloxacin, 2.0 to 2.8 mg/kg). In conclusion, linezolid does not seem to potentiate infections caused by Gram-negative pathogens or to interact antagonistically with ciprofloxacin.

The Contribution of Genomics to the Discovery of New Antibiotics

The emergence of common bacterial pathogens that are resistant to multiple antibiotics, coupled with the failure of traditional methods to yield new anti-infective agents, threatens current paradigms of therapeutic intervention (Omura, 1992; Shlaes et al., 1991; Tenover and Hughes, 1996). The current focus has been on improving existing antibiotic classes while little progress has been made in discovering chemically novel anti-infective agents. This unmet medical need may now be addressed if we can successfully exploit the new wealth of genomic sequence data to devise novel strategies for drug discovery (Moir et al., 1999). Whole genome comparative sequence analysis now allows the identification of genes/gene products that are common to many or all pathogenic bacteria of clinical importance. bioinformatics-based gene homology and motif analyses allow rapid phylogenetic comparisons to be made and, in addition, predict functional information critical to target selection. Putative targets can then be assessed rapidly using gene essentiality testing methods which allow analyses both in vitro and in models of the infection state. Sensitive and direct in vivo expression analyses confirm that the expression of the target gene is relevant to the establishment and maintenance of infection. Ultimately, the gene products must be screened against novel chemical libraries and natural product banks derived from a wide bio-diversity in order to identify lead compounds with potential antibiotic activities that will be developed to provide the next generation of therapeutic agents. Those companies which can adapt and implement these genomic-based technologies, derive significant competitive advantage in creating product portfolios that will address the unmet clinical need.