Francis Tally and the discovery and development of tigecycline: a personal reminiscence.

For Francis Tally, both medicine and science were highly personal undertakings. Tally thought that emotional engagement was important in one's work and one's life, which were inseparable in his case. Indeed, Tally materially participated in no fewer than 4 programs that resulted in the approval and commercialization of novel antibiotics. These included piperacillin-tazobactam (which is currently the injectable antibiotic with the largest volume of sales worldwide) and daptomycin. This article focuses on the discovery and development of tigecycline.

Establishment of in vitro susceptibility testing methodologies and comparative activities of piperacillin in combination with the penem {beta}-lactamase inhibitor BLI-489.

The novel bicyclic penem inhibitor BLI-489 has demonstrated activity as an inhibitor of class A, C, and D beta-lactamases. To determine the combination of piperacillin and BLI-489 to be used in susceptibility testing that would most accurately identify susceptible and resistant isolates, a predictor panel of beta-lactamase-producing bacteria was utilized to determine the reliability of the combination of piperacillin-BLI-489 at a constant inhibitor concentration of 2 or 4 microg/ml and at ratios of 1:1, 2:1, 4:1, and 8:1. There were a number of strains that would be falsely reported as susceptible or intermediate if tested with the ratios of 1:1 and 2:1, whereas the constant concentration of 2 microg/ml of BLI-489 and the ratio of 8:1 had a tendency to overpredict resistance. Similar MICs were obtained with piperacillin-BLI-489 in a 4:1 ratio and when BLI-489 was held constant at 4 microg/ml. Based on these results, an in vitro testing methodology employing a constant concentration of 4 microg/ml BLI-489 was used to evaluate the combination of piperacillin-BLI-489 against a larger panel of recently identified clinical isolates. Approximately 55% of all of the enteric bacilli tested were nonsusceptible to piperacillin alone (MIC > or = 32 microg/ml). However, 92% of these piperacillin nonsusceptible strains were inhibited by < or =16 microg/ml piperacillin-BLI-489; in contrast, only 66% were inhibited by < or =16 microg/ml piperacillin-tazobactam. The combination of piperacillin-BLI-489 also demonstrated improved activity compared to that of piperacillin-tazobactam against the problematic extended-spectrum beta-lactamase- and AmpC-expressing strains.

Late stage antibacterial drugs in the clinical pipeline.

Bacterial resistance to antimicrobial agents is a growing problem worldwide. Not only is issue compounded by the fact that there are fewer pharmaceutical companies conducting research to discover novel antimicrobials than in the past but development time lines have stretched so that a dozen years from discovery to the market is now the standard. Eleven antibacterial drugs in late stage clinical development are discussed. Whereas many of these may successfully deal with resistant strains of Gram-positive pathogens, there is very little in development to address the gorwing unmet medical need of multi-drug resistant Gram-negative infections.

Staphylococcal vaccines and immunotherapy: to dream the impossible dream?

Staphylococcal infections not only remain an important cause of morbidity and mortality in both the community and the clinic, but the emergence of a global pandemic of community-associated methicillin-resistant Staphylococcus aureus, involving what purports to be a more virulent strain of this organism, has also led several in the infectious disease community to call for improved disease prevention strategies (in addition to novel therapeutics) in what could be thought of as the microbiological version of pre-emption. In this case, Staphylococcus aureus possesses "weapons of mass destruction" and appears to be using them effectively as part of anti-immunization insurgency.

Why is big Pharma getting out of antibacterial drug discovery?

Since the advent of the antibiotic era in the late 1940s drug discovery and development has evolved into an expensive, time consuming, cumbersome and bureaucratic process involving multiple interest groups such as pharmaceutical manufacturers, governmental regulatory authorities, patent officers, academic and clinical researchers and trial lawyers. It would seem that the least involved among the interest groups are the consumers of health care themselves. Politicians and the public alike complain loudly about drug prices although fewer and fewer new therapies are being developed. The cost and complexities of drug discovery and development have shifted the investment equation away from the development of drugs targeting short course therapies for acute diseases and towards long-term treatment of chronic conditions. Coupled with the failure of large investments into target-based approaches to produce novel antibacterial agents, companies large and small have exited from this field despite a growing clinical need.

Inactivation of mprF affects vancomycin susceptibility in Staphylococcus aureus.

A chemically generated mutant of Staphylococcus aureus RN4220, GC6668, was isolated that had a fourfold increase in resistance to vancomycin. This phenotype reverted back to susceptibility by insertional mutagenesis with Tn917. In a selected set of revertants, Tn917 insertion was mapped to a unique chromosomal region upstream of mprF, a recently described gene that determines staphylococcal resistance to several host defense peptides. The genetic linkage between the vancomycin susceptibility and Tn917 insertion was then confirmed by transduction backcrosses into both GC6668 and GISA isolates, MER-S12 and HT2002 0127. Northern blot analysis, insertional inactivation and complementation experiments showed that mprF mediates vancomycin susceptibility in S. aureus. The inactivation of mprF by Tn917 insertion in HT2002 0127 caused a significant increase in the binding of vancomycin to the cell membranes. This observation serves as a likely mechanism of the increased vancomycin susceptibility associated with mprF inactivation.

New (and not so new) antibacterial targets - from where and when will the novel drugs come?

Although the need for new antibacterial therapies and strategies is greater than it has been in a quarter of a century and despite considerable effort, little progress has been observed in the development of agents. Target-based screening for new antibacterial agents has been particularly disappointing, despite a plethora of potential targets, enhanced screening methodologies and considerable investment. Recent efforts are coalescing around tried and true biosynthetic pathways like cell wall biosynthesis and the less well exploited pathway for fatty acid biosynthesis. Novel crystal structures for components of the replication complex and the ribosome have fuelled efforts at screening for novel replication and translation inhibitors. And target-based screening appears to have scored at least a modest success with inhibitors of peptide deformylase. More problematic are strategies targeting virulence and regulation of gene expression; despite considerably better understanding of these processes, there is no clear path to the use of inhibitors of host-pathogen interactions and/or regulation. In targeting the expression of resistance itself, it appears that staying one step ahead of the beta-lactamases is an overly optimistic goal; the best that can be expected is to avoid falling too far behind. Targeting multidrug efflux pumps may be more edifying in the long run.

Genomics in anti-infective drug discovery--getting to endgame.

Whole chromosome sequence of prokaryotes has provided the availability of multiple bacterial pathogen sequence data and it has become a widely used tool in the drug discovery process. However the sequence data in itself is merely a starting point for drug discovery of novel antibacterial targets and, eventually, drugs. In order to leverage this large amount of data it is necessary to match an understanding of the microbial physiology of pathogenic bacteria to disease processes and identifying the gene products for which intervention may reduce or eliminate the infectious state. However, to date, the application of genomics to anti-infective drug discovery has not, since 1995 with the first complete sequence of a pathogenic bacterium, led to identification of a novel antibacterial agent. Here we review the field of bacterial genomics and how it is enabling the drug discovery process. Many new molecular-based technologies (proteomics, transcriptional profiling, studies of gene expression in vivo) have originated or have expanded into wider use, and have been made possible by the availability of complete bacterial genome sequence information and subsequent bioinformatic analytic tools. Taken together, these technologies, overlaid within an established drug discovery program, now affords the opportunity for the identification, validation, and process design for high-throughput target mining at unprecedented volumes and timeframes.

Hydrophobic acetal and ketal derivatives of mannopeptimycin-alpha and desmethylhexahydromannopeptimycin-alpha: semisynthetic glycopeptides with potent activity against Gram-positive bacteria.

The effect of introducing hydrophobic groups onto the disaccharide portion of the mannopeptimycins has been examined. Under acid-catalyzed conditions dimethyl acetals and ketals react on the terminal mannose of the disaccharide moiety of mannopeptimycin-alpha and the cyclohexylalanyl analogue 2. The preferentially formed monofunctionalized 4,6-acetals and -ketals display potent antibacterial activities against Gram-positive microorganisms, including MRSA, PRSP, and VRE pathogens.

Recurrent episodes of shock-like syndrome caused by the same strain of vancomycin-resistant Enterococcus faecium in a pediatric patient.

We present the case of a hospitalized pediatric patient with short bowel syndrome who was dependent upon total parenteral nutrition for 17 months. Shortly after admission she became colonized with vancomycin-resistant Enterococcus faecium (VRE) and developed 12 distinct episodes of serious infection associated with it. The course of VRE colonization and infections in this patient was studied through analysis of 40 representative isolates obtained from different sites during distinct episodes of infection. Standard microbiological techniques, automated ribosomal DNA typing, polymerase chain reaction, and pulsed-field gel electrophoresis (PFGE) were used. All isolates except for the one associated with the initial episode of bacteremia were VRE, and were multidrug resistant. The last four episodes of infection were caused by isolates resistant to all tested antibiotics except for intermediate susceptibility to chloramphenicol. The vanA genotype was a source of vancomycin resistance in all VRE isolates. Both ribotyping and PFGE showed two distinct clones of VRE in clinical and stool surveillance isolates: one was associated with clinical illness and the other was not associated with infection. Recurrent VRE infections occur as a consequence of prolonged gastrointestinal colonization. Morbidity is associated with host factors, the presence of co-pathogens, and possibly intrinsically more virulent VRE strain.

Characterization of the Staphylococcus aureus CidR regulon: elucidation of a novel role for acetoin metabolism in cell death and lysis.

The Staphylococcus aureus cid and lrg operons encode a novel regulatory system that affects murein hydrolase activity, stationary-phase survival and antibiotic tolerance. Expression of the lrgAB operon is positively regulated by a two-component regulatory system encoded by the lytSR operon located immediately upstream to lrgAB. By comparison, the cidABC operon lies downstream from the cidR gene, encoding a protein homologous to the LysR-type family of transcriptional regulators. Transcription analysis of a cidR mutant revealed that CidR enhances cidABC expression in the presence of acetic acid generated by the metabolism of excess glucose. Microarray studies identified additional CidR-regulated operons including the IrgAB and alsSD encoding proteins involved in acetoin production. Surprisingly, Northern blot analyses revealed that cidABC and lrgAB transcription was uninducible in an alsSD mutant grown in the presence of excess glucose, suggesting that the CidR-mediated upregulation of cidABC and lrgAB transcription is dependent on the presence of intact alsSD genes. Zymographic and quantitative analyses of murein hydrolase activity also revealed that disruption of the alsSD genes results in significantly decreased extracellular murein hydrolase activity compared with that of the parental strain, UAMS-1. Furthermore, the alsSD mutant displayed decreased stationary-phase survival relative to UAMS-1, both in the presence and absence of glucose. The results of this study define the CidR regulon and demonstrate that the generation of acetoin is linked to the control of cell death and lysis in S. aureus.

Transcription Profiling of the mgrA Regulon in Staphylococcus aureus.

MgrA has been shown to affect multiple Staphylococcus aureus genes involved in virulence and antibiotic resistance. To comprehensively identify the target genes regulated by mgrA, we employed a microarray method to analyze the transcription profiles of S. aureus Newman, its isogeneic mgrA mutant, and an MgrA-overproducing derivative. We compared genes that were differentially expressed at exponential or early stationary growth phases. Our results showed that MgrA affected an impressive number of genes, 175 of which were positively regulated and 180 of which were negatively regulated in an mgrA-specific manner. The target genes included all functional categories. The microarray results were validated by real-time reverse transcription-PCR quantitation of a set of selected genes from different functional categories. Our data also indicate that mgrA regulates virulence factors in a fashion analogous to that of the accessory gene regulatory locus (agr). Accordingly, exoproteins are upregulated and surface proteins are downregulated by the regulator, suggesting that mgrA may function in concert with agr. The fact that a large number of genes are regulated by mgrA implies that MgrA is a major global regulator in S. aureus.

Characterization of a strain of community-associated methicillin-resistant Staphylococcus aureus widely disseminated in the United States.

A highly stable strain of Staphylococcus aureus with a pulsed-field gel electrophoresis type of USA300 and multilocus sequence type 8 has been isolated from patients residing in diverse geographic regions of the United States. This strain, designated USA300-0114, is a major cause of skin and soft tissue infections among persons in community settings, including day care centers and correctional facilities, and among sports teams, Native Americans, men who have sex with men, and military recruits. The organism is typically resistant to penicillin, oxacillin, and erythromycin (the latter mediated by msrA) and carries SCCmec type IVa. This strain is variably resistant to tetracycline [mediated by tet(K)]; several recent isolates have decreased susceptibility to fluoroquinolones. S. aureus USA300-0114 harbors the genes encoding the Panton-Valentine leucocidin toxin. DNA sequence analysis of the direct repeat units within the mec determinant of 30 USA300-0114 isolates revealed differences in only a single isolate. Plasmid analysis identified a common 30-kb plasmid that hybridized with blaZ and msrA probes and a 3.1-kb cryptic plasmid. A 4.3-kb plasmid encoding tet(K) and a 2.6-kb plasmid encoding ermC were observed in a few isolates. DNA microarray analysis was used to determine the genetic loci for a series of virulence factors and genes associated with antimicrobial resistance. Comparative genomics between USA300-0114 and three other S. aureus lineages (USA100, USA400, and USA500) defined a set of USA300-0114-specific genes, which may facilitate the strain's pathogenesis within diverse environments.

Characterization of the Staphylococcus aureus heat shock, cold shock, stringent, and SOS responses and their effects on log-phase mRNA turnover.

Despite its being a leading cause of nosocomal and community-acquired infections, surprisingly little is known about Staphylococcus aureus stress responses. In the current study, Affymetrix S. aureus GeneChips were used to define transcriptome changes in response to cold shock, heat shock, stringent, and SOS response-inducing conditions. Additionally, the RNA turnover properties of each response were measured. Each stress response induced distinct biological processes, subsets of virulence factors, and antibiotic determinants. The results were validated by real-time PCR and stress-mediated changes in antimicrobial agent susceptibility. Collectively, many S. aureus stress-responsive functions are conserved across bacteria, whereas others are unique to the organism. Sets of small stable RNA molecules with no open reading frames were also components of each response. Induction of the stringent, cold shock, and heat shock responses dramatically stabilized most mRNA species. Correlations between mRNA turnover properties and transcript titers suggest that S. aureus stress response-dependent alterations in transcript abundances can, in part, be attributed to alterations in RNA stability. This phenomenon was not observed within SOS-responsive cells.

Transcriptional profiling of a Staphylococcus aureus clinical isolate and its isogenic agr and sarA mutants reveals global differences in comparison to the laboratory strain RN6390.

The production of Staphylococcus aureus virulence factors is under the control of complex regulatory circuits. Most studies aimed at defining these regulatory networks have focused on derivatives of the strain NCTC 8325, most notably RN6390. However, all NCTC 8325 derivatives, including RN6390, possess an 11 bp deletion in rsbU. This deletion renders NCTC 8325 derivatives naturally sigma-factor-B deficient. Recent studies have shown that RN6390 is also deficient, in comparison to clinical isolates, with respect to biofilm formation, a process which is important for both pathogenesis and antimicrobial resistance. Based on these considerations, the authors carried out genome-scale transcriptional profiling, comparing RN6390 with the virulent rsbU-positive clinical isolate UAMS-1. The results revealed significant genome-wide differences in expression patterns between RN6390 and UAMS-1, and suggested that the overall transcriptional profile of UAMS-1 is geared toward expression of factors that promote colonization and biofilm formation. In contrast, the transcriptional profile of RN6390 was heavily influenced by RNAIII expression, resulting in a phenotype characterized by increased production of exoproteins, and decreased capacity to form a biofilm. The greater influence of agr in RN6390 relative to UAMS-1 was also evident when the transcriptional profile of UAMS-1 was compared with that of its isogenic sarA and agr mutants. Specifically, the results indicate that, in contrast to NCTC 8325 derivatives, agr plays a limited role in overall regulation of gene expression in UAMS-1, when compared with sarA. Furthermore, by defining the sarA regulon in a biofilm-positive clinical isolate, and comparing the results with transcriptional profiling experiments defining biofilm-associated gene expression patterns in the same strain, the authors identified a sarA-regulated operon (alsSD) that is also induced in biofilms, and demonstrated that mutation of alsSD results in reduced capacity to form a biofilm.

Characterizing the effect of the Staphylococcus aureus virulence factor regulator, SarA, on log-phase mRNA half-lives.

Bacterial pathogens regulate virulence factor expression at both the level of transcription initiation and mRNA processing/turnover. Within Staphylococcus aureus, virulence factor transcript synthesis is regulated by a number of two-component regulatory systems, the DNA binding protein SarA, and the SarA family of homologues. However, little is known about the factors that modulate mRNA stability or influence transcript degradation within the organism. As our entree to characterizing these processes, S. aureus GeneChips were used to simultaneously determine the mRNA half-lives of all transcripts produced during log-phase growth. It was found that the majority of log-phase transcripts (90%) have a short half-life (<5 min), whereas others are more stable, suggesting that cis- and/or trans-acting factors influence S. aureus mRNA stability. In support of this, it was found that two virulence factor transcripts, cna and spa, were stabilized in a sarA-dependent manner. These results were validated by complementation and real-time PCR and suggest that SarA may regulate target gene expression in a previously unrecognized manner by posttranscriptionally modulating mRNA turnover. Additionally, it was found that S. aureus produces a set of stable RNA molecules with no predicted open reading frame. Based on the importance of the S. aureus agr RNA molecule, RNAIII, and small stable RNA molecules within other pathogens, it is possible that these RNA molecules influence biological processes within the organism.

A novel MATE family efflux pump contributes to the reduced susceptibility of laboratory-derived Staphylococcus aureus mutants to tigecycline.

Tigecycline, an expanded-broad-spectrum glycylcycline antibiotic is not affected by the classical tetracycline resistance determinants found in Staphylococcus aureus. The in vitro selection of mutants with reduced susceptibility to tigecycline was evaluated for two methicillin-resistant S. aureus strains by serial passage in increasing concentrations of tigecycline. Both strains showed a stepwise elevation in tigecycline MIC over a period of 16 days, resulting in an increase in tigecycline MIC of 16- and 32-fold for N315 and Mu3, respectively. Transcriptional profiling revealed that both mutants exhibited over 100-fold increased expression of a gene cluster, mepRAB (multidrug export protein), encoding a MarR-like transcriptional regulator (mepR), a novel MATE family efflux pump (mepA), and a hypothetical protein of unknown function (mepB). Sequencing of the mepR gene in the mutant strains identified changes that presumably inactivated the MepR protein, which suggested that MepR functions as a repressor of mepA. Overexpression of mepA in a wild-type background caused a decrease in susceptibility to tigecycline and other substrates for MATE-type efflux pumps, although it was not sufficient to confer high-level resistance to tigecycline. Complementation of the mepR defect by overexpressing a wild-type mepR gene reduced mepA transcription and lowered the tigecycline MIC in the mutants. Transcription of tet(M) also increased by over 40-fold in the Mu3 mutant. This was attributed to a deletion in the promoter region of the gene that removed a stem-loop responsible for transcriptional attenuation. However, overexpression of the tet(M) transcript in a tigecycline-susceptible strain was not enough to significantly increase the MIC of tigecycline. These results suggest that the overexpression of mepA but not tet(M) may contribute to decreased susceptibility of tigecycline in S. aureus.

Comparative genomics of Staphylococcus aureus musculoskeletal isolates.

Much of the research aimed at defining the pathogenesis of Staphylococcus aureus has been done with a limited number of strains, most notably the 8325-4 derivative RN6390. Several lines of evidence indicate that this strain is unique by comparison to clinical isolates of S. aureus. Based on this, we have focused our efforts on two clinical isolates (UAMS-1 and UAMS-601), both of which are hypervirulent in our animal models of musculoskeletal infection. In this study, we used comparative genomic hybridization to assess the genome content of these two isolates relative to RN6390 and each of seven sequenced S. aureus isolates. Our comparisons were done by using an amplicon-based microarray from the Pathogen Functional Genomics Resource Center and an Affymetrix GeneChip that collectively represent the genomes of all seven sequenced strains. Our results confirmed that UAMS-1 and UAMS-601 share specific attributes that distinguish them from RN6390. Potentially important differences included the presence of cna and the absence of isaB, sarT, sarU, and sasG in the UAMS isolates. Among the sequenced strains, the UAMS isolates were most closely related to the dominant European clone EMRSA-16. In contrast, RN6390, NCTC 8325, and COL formed a distinct cluster that, by comparison to the other four sequenced strains (Mu50, N315, MW2, and SANGER-476), was the most distantly related to the UAMS isolates and EMRSA-16.