Biomedical subjects

S J Projan

Publications and source records attributed to S J Projan.

At least 19 recordsLinked to original sources

PubMed2005

MgrA is a multiple regulator of two new efflux pumps in Staphylococcus aureus.

In an analysis of the resistance mechanisms of an mgrA mutant, we identified two genes encoding previously undescribed transporters, NorB and Tet38. norB was 1,392 bp and encoded a predicted 49-kDa protein. When overexpressed, NorB led to an increase in resistance to hydrophilic quinolones, ethidium bromide, and cetrimide and also to sparfloxacin, moxifloxacin, and tetracycline, a resistance phenotype of the mgrA mutant. NorA and NorB shared 30% similarity, and NorB shared 30 and 41% similarities with the Bmr and Blt transporters of Bacillus subtilis, respectively. The second efflux pump was a more selective transporter that we have called Tet38, which had 46% similarity with the plasmid-encoded TetK efflux transporter of S. aureus. tet38 was 1,353 bp and encoded a predicted 49-kDa protein. Overexpression of tet38 produced resistance to tetracycline but not to minocycline and other drugs. norB and tet38 transcription was negatively regulated by MgrA. Limited binding of MgrA to the promoter regions of norB and tet38 was demonstrated by gel shift assays, suggesting that MgrA was an indirect regulator of norB and tet38 expression. The mgrA norB double mutant was reproducibly twofold more susceptible to the tested quinolones than the mgrA mutant. The mgrA tet38 double mutant became more susceptible to tetracycline than the wild-type parent strain. These data demonstrate that overexpression of NorB and Tet38 contribute, respectively, to the hydrophobic quinolone resistance and the tetracycline resistance of the mgrA mutant and that MgrA regulates expression of norB and tet38 in addition to its role in regulation of norA expression.

Amino Acid Sequence↗

PubMed2004

Antibacterial drug discovery: is it all downhill from here?

There has been a marked decline in the industrial research aimed at discovering novel antibacterial agents, including new drugs that target resistant organisms. While this decline may reflect past cyclical changes that often affect resource allocation at pharmaceutical companies, this decline is occurring at a time of increasing levels of antibacterial drug resistance and meagre pipelines of new agents that are active against them. There are multiple reasons for this decline, although few are unique to antibacterial drug discovery research. These include: lack of industry productivity, increasing size of clinical trials, increased generic competition and other pressures on drug pricing, a crowded and confused marketplace and industry consolidation. And while many (if not most) large companies and biotechs have exited the field or severely curtailed their research, others have made it a point to continue their efforts, citing both the unmet medical need and a large and apparently growing market. Despite the fact that some companies have remained engaged, the view here is that the current level of industrial effort is insufficient to sustain a healthy flow of new and better agents that are needed to counter the imminent threat of bacterial drug resistance. Therefore, a clear and urgent need for finding ways to improve the level and quality of industrial research in this area is apparent.

Anti-Bacterial Agents↗

PubMed2004

Uses of Staphylococcus aureus GeneChips in genotyping and genetic composition analysis.

Understanding the relatedness of strains within a bacterial species is essential for monitoring reservoirs of antimicrobial resistance and for epidemiological studies. Pulsed-field gel electrophoresis (PFGE), ribotyping, and multilocus sequence typing are commonly used for this purpose. However, these techniques are either nonquantitative or provide only a limited estimation of strain relatedness. Moreover, they cannot extensively define the genes that constitute an organism. In the present study, 21 oxacillin-resistant Staphylococcus aureus (ORSA) isolates, representing eight major ORSA lineages, and each of the seven strains for which the complete genomic sequence is publicly available were genotyped using a novel GeneChip-based approach. Strains were also subjected to PFGE and ribotyping analysis. GeneChip results provided a higher level of discrimination among isolates than either ribotyping or PFGE, although strain clustering was similar among the three techniques. In addition, GeneChip signal intensity cutoff values were empirically determined to provide extensive data on the genetic composition of each isolate analyzed. Using this technology it was shown that strains could be examined for each element represented on the GeneChip, including virulence factors, antimicrobial resistance determinants, and agr type. These results were validated by PCR, growth on selective media, and detailed in silico analysis of each of the sequenced genomes. Collectively, this work demonstrates that GeneChips provide extensive genotyping information for S. aureus strains and may play a major role in epidemiological studies in the future where correlating genes with particular disease phenotypes is critical.

Algorithms↗

PubMed2003

Genome-wide transcriptional profiling of the response of Staphylococcus aureus to cell-wall-active antibiotics reveals a cell-wall-stress stimulon.

The molecular events following inhibition of bacterial peptidoglycan synthesis have not been studied extensively. Previous proteomic studies have revealed that certain proteins are produced in increased amounts upon challenge of Staphylococcus aureus with cell-wall-active antibiotics. In an effort to further those studies, the genes upregulated in their expression in response to cell-wall-active antibiotics have been identified by genome-wide transcriptional profiling using custom-made Affymetrix S. aureus GeneChips. A large number of genes, including ones encoding proteins involved in cell-wall metabolism (including pbpB, murZ, fmt and vraS) and stress responses (including msrA, htrA, psrA and hslO), were upregulated by oxacillin, D-cycloserine or bacitracin. This response may represent the transcriptional signature of a cell-wall stimulon induced in response to cell-wall-active agents. The findings imply that treatment with cell-wall-active antibiotics results in damage to proteins including oxidative damage. Additional genes in a variety of functional categories were upregulated uniquely by each of the three cell-wall-active antibiotics studied. These changes in gene expression can be viewed as an attempt by the organism to defend itself against the antibacterial activities of the agents.

Anti-Bacterial Agents↗

PubMed2003

Global regulation of Staphylococcus aureus genes by Rot.

Staphylococcus aureus produces a wide array of cell surface and extracellular proteins involved in virulence. Expression of these virulence factors is tightly controlled by numerous regulatory loci, including agr, sar, sigB, sae, and arl, as well as by a number of proteins with homology to SarA. Rot (repressor of toxins), a SarA homologue, was previously identified in a library of transposon-induced mutants created in an agr-negative strain by screening for restored protease and alpha-toxin. To date, all of the SarA homologues have been shown to act as global regulators of virulence genes. Therefore, we investigated the extent of transcriptional regulation of staphylococcal genes by Rot. We compared the transcriptional profile of a rot agr double mutant to that of its agr parental strain by using custom-made Affymetrix GeneChips. Our findings indicate that Rot is not only a repressor but a global regulator with both positive and negative effects on the expression of S. aureus genes. Our data also indicate that Rot and agr have opposing effects on select target genes. These results provide further insight into the role of Rot in the regulatory cascade of S. aureus virulence gene expression.

Bacterial Proteins↗

PubMed2001

Antibiotic resistance in bacteria from magpies (Pica pica) and rabbits (Oryctolagus cuniculus) from west Wales.

The prevalence of antibiotic-resistant bacteria in wild animal and bird populations is largely unknown, with little consistency among the few published reports. We therefore examined intestinal bacteria from magpies (Pica pica) and rabbits (Oryctolagus cuniculus) collected in rural west Wales. Escherichia coli isolates resistant to multiple antibiotics were grown from eight of 20 magpies trapped in spring, 1999 and one of 17 in spring, 2000; the most prevalent resistance trait among these isolates was to tetracycline, but resistances to ampicillin, chloramphenicol, kanamycin, sulphonamide, tetracycline and trimethoprim were also found. Tetracycline-resistant Enterococcus spp. were found in one of 20 magpies in 1999 and three of 17 in 2000. Only one resistant E. coli isolate was detected among gut bacteria from 13 rabbits, and this strain was resistant only to tetracycline. Differences in the prevalence of resistance between bacteria from rabbits and magpies may reflect differences in diet: rabbits graze field edges, whereas magpies are omnivorous and opportunistic. The resistance genes found in E. coli isolates from magpies mostly corresponded to those common among human isolates, but those conferring tetracycline resistance were unique.

Animals↗

PubMed2001

Transcription profiling-based identification of Staphylococcus aureus genes regulated by the agr and/or sarA loci.

The advent of transcription profiling technologies has provided researchers with an unprecedented ability to study biological processes. Accordingly, a custom-made Affymetrix GeneChip, constituting >86% of the Staphylococcus aureus genome, was used to identify open reading frames that are regulated by agr and/or SarA, the two best-studied regulators of the organism's virulence response. RNA extracted from wild-type cells and agr, sarA, and agr sarA mutant cells in the early-, mid-, and late-log and stationary phases of growth was analyzed. Open reading frames with transcription patterns expected of genes either up- or downregulated in an agr- and/or SarA-dependent manner were identified. Oligonucleotide microarray and Northern blot analyses confirmed that the transcription of several known virulence genes, including hla (alpha-toxin) and spa (protein A), is regulated by each effector and provided insights about the regulatory cascades involved in both alpha-hemolysin and protein A expression. Several putative virulence factors were also identified as regulated by agr and/or SarA. In addition, genes that are involved in several biological processes but which are difficult to reconcile as playing a direct role in the organism's pathogenesis also appeared to be regulated by each effector, suggesting that products of both the agr and the sarA locus are more-global transcription regulators than previously realized.

Bacterial Proteins↗

PubMed2000

Bacterial virulence as a target for antimicrobial chemotherapy.

As bacterial resistance to currently used antibiotics increases, so too must efforts to identify novel agents and strategies for the prevention and treatment of bacterial infection. In the past, antimicrobial drug discovery efforts have focused on eradicating infection by either cidal or static agents, resulting in clearance of the bacterium from the infected host. To this end, drug discovery targets have been those proteins or processes essential for bacterial cell viability. However, inhibition of the interaction between the bacterium and its host may also be a target. During establishment of an infection, pathogenic bacteria use carefully regulated pathways of conditional gene expression to transition from a free-living form to one that must adapt to the host milieu. This transition requires the regulated production of both extracellular and cell-surface molecules, often termed virulence factors. As the biological imperatives of the invading organism change during the course of an infection, the expression of these factors is altered in response to environmental cues. These may be changes in the host environment, for example, pH, metabolites, metal ions, osmolarity, and temperature. Alternatively, effector molecules produced by the bacterium to sense changing cell density can also lead to changes in virulence gene expression. Although the mechanisms of pathogenesis among different bacteria vary, the principles of virulence are generally conserved. Bacterial virulence may therefore offer unique opportunities to inhibit the establishment of infection or alter its course as a method of antimicrobial chemotherapy.

Anti-Bacterial Agents↗

PubMed2000

Mutations in the interdomain loop region of the tetA(A) tetracycline resistance gene increase efflux of minocycline and glycylcyclines.

A novel class of tetracyclines, the glycylcyclines, have been shown to be active against bacterial strains harboring genes encoding tetracycline efflux pumps. However, two veterinary Salmonella isolates that carried tetracycline resistance determinants of the tetA(A) class were found to have reduced susceptibility to glycylcyclines, especially two early investigational glycylcyclines, DMG-MINO and DMG-DMDOT. These isolates were also quite resistant to tetracycline and minocycline. The isolates, one a strain of S. cholerasuis and the other, S. typhimurium, both carried the same novel tetA(A) variant, based on DNA sequencing, with one determinant plasmid encoded and the other located on the chromosome. This tetA(A) variant was cloned and shown to provide reduced susceptibility to the glycylcycline class although GAR-936, a glycylcycline currently in clinical development, was the least affected. The novel tetA(A) gene carries two mutations in the largest cytoplasmic loop of the efflux pump, which causes a double frameshift in codons 201, 202, and 203. This "interdomain region" of the efflux pump has generally been regarded as having no functional role in the efflux of tetracycline but the double frameshift is most likely responsible for the enhanced resistance observed and points to an interaction that was previously unrecognized. Mutants of the tetA(B) class with decreased susceptibility to the glycylcyclines were also generated in vitro. These all carried mutations in the portion of the tetA(B) gene encoding a transmembrane spanning region of the efflux pump. The laboratory-generated mutants point to the tight constraints in substrate recognition of the transmembrane-spanning region and may suggest that it will be the interdomain region of the pump that is likely to be the locus of future glycylcycline resistance mutations as these compounds enter clinical use.

Amino Acid Sequence↗

PubMed2000

Identification and analysis of bacterial protein secretion inhibitors utilizing a SecA-LacZ reporter fusion system.

Protein secretion is an essential process for bacterial growth, yet there are few if any antimicrobial agents which inhibit secretion. An in vivo, high-throughput screen to detect secretion inhibitors was developed based on the translational autoregulation of one of the central protein components, SecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escherichia coli which is induced when secretion is perturbed. Several compounds, including two natural product extracts, which had the ability to induce the reporter fusion were identified and the MICs of these compounds for Staphylococcus aureus strain MN8 were found to be < or =128 microg/ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques were used to analyze the affects of these compounds on protein secretion. Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity.

Adenosine Triphosphatases↗

PubMed2000

Preclinical pharmacology of GAR-936, a novel glycylcycline antibacterial agent.

GAR-936 is an analog of minocycline, a semisynthetic derivative of tetracycline. It has broad-spectrum antibacterial activity in vitro and in vivo. The new class of tetracyclines to which GAR-936 belongs is named the glycylcyclines. Tetracyclines act by inhibiting protein translation in bacteria, presumably by binding to the 30S ribosomal subunit and blocking entry of amino-acyl transfer RNA molecules into the A site of the ribosome. This prevents incorporation of amino acid residues into elongating peptide chains. In general, tetracyclines are considered bacteriostatic and the critical therapeutic parameter is the area under the concentration-time curve. GAR-936 has bactericidal activity; at 4 times the minimum inhibitory concentration, a 2- to 3-log reduction in colony counts was seen against Streptococcus pneumoniae, Neisseria gonorrhoeae, Haemophilus influenzae, Escherichia coli, and Staphylococcus aureus. GAR-936 is active against the antibiotic-resistant gram-positive bacteria methicillin-resistant Staphylococcus aureus, penicillin-resistant S. pneumoniae, and vancomycin-resistant enterococci. It is most significant that GAR-936 and other glycylcyclines are active against bacterial strains carrying either or both of the two major forms of tetracycline resistance: efflux and ribosomal protection. Using isogenic panels of bacteria carrying various tetracycline-resistance determinants, a series of more than 300 analogs was tested for antibacterial activity, which allowed for structure-activity relationships to be determined. Results indicated that certain substituents at the 9 position of the tetracycline molecule restored activity against bacteria harboring genes encoding either or both efflux and ribosomal protection. A single chemical modification overcame the two molecularly distinct forms of resistance while maintaining activity against susceptible gram-positive, gram-negative, aerobic, and anaerobic bacteria. Although mutants can be generated that are less susceptible to previously studied glycylcyclines, only marginal differences in susceptibility to GAR-936 were noted. Therefore, whereas emergence of resistance to any widely administered antibiotic is a foregone conclusion, resistance to GAR-936 will not readily arise by trivial mutations in existing resistance genes.

Anti-Bacterial Agents↗

PubMed1999

SarA, a global regulator of virulence determinants in Staphylococcus aureus, binds to a conserved motif essential for sar-dependent gene regulation.

The expression of many virulence determinants in Staphylococcus aureus including alpha-hemolysin-, protein A-, and fibronectin-binding proteins is controlled by global regulatory loci such as sar and agr. In addition to controlling target gene expression via agr (e.g. alpha-hemolysin), the sar locus can also regulate target gene transcription via agr-independent mechanisms. In particular, we have found that SarA, the major regulatory protein encoded within sar, binds to a conserved sequence, homologous to the SarA-binding site on the agr promoter, upstream of the -35 promoter boxes of several target genes including hla (alpha-hemolysin gene), spa (protein A gene), fnb (fibronectin-binding protein genes), and sec (enterotoxin C gene). Deletion of the SarA recognition motif in the promoter regions of agr and hla in shuttle plasmids rendered the transcription of these genes undetectable in agr and hla mutants, respectively. Likewise, the transcription activity of spa (a gene normally repressed by sar), as measured by a XylE reporter fusion assay, became derepressed in a wild type strain containing a shuttle plasmid in which the SarA recognition site had been deleted from the spa promoter region. However, DNase I footprinting assays demonstrated that the SarA-binding region on the spa and hla promoter is more extensive than the predicted consensus sequence, thus raising the possibility that the consensus sequence is an activation site within a larger binding region. Because the sar and agr regulate an assortment of virulence factors in S. aureus, we propose, based on our data, a unifying hypothesis for virulence gene activation in S. aureus whereby SarA is a regulatory protein that binds to its consensus SarA recognition motif to activate (e.g. hla) or repress (e.g. spa) the transcription of sar target genes, thus accounting for both agr-dependent and agr-independent mode of regulation.

Bacterial Proteins↗

PubMed1999

Clinical characteristics and molecular epidemiology associated with imipenem-resistant Klebsiella pneumoniae.

Eight patients were infected or colonized with imipenem-resistant Klebsiella pneumoniae (IRKP) from December 1994 to November 1995. Initial Klebsiella isolates were susceptible to imipenem but resistant to all cephalosporins, aminoglycosides, and beta-lactam inhibitor combinations. All patients had been in the surgical intensive care unit and had undergone abdominal surgery or tracheostomy during hospitalization. The average age of the patients was 71 years (range, 41-81 years). All patients were treated with imipenem for 5 to 36 days, and IRKP was recovered from each during or after therapy. Pulsed-field gel electrophoresis (PFGE) of the IRKP isolates revealed three distinct clonal patterns. Paired sequential isolates of imipenem-susceptible K. pneumoniae and IRKP from two patients had identical PFGE patterns, suggesting the development of clonal stepwise resistance to imipenem during therapy. Thus, imipenem resistance in Klebsiella may occur when this agent is used for treatment of infection due to ceftazidine- and aminoglycoside-resistant strains.

Adult↗

PubMed1998

Recent developments in tetracycline antibiotics.

The rapid emergence of pathogenic bacteria resistant to tetracyclines and other currently available antibiotics has caused serious concern among medical professionals. It has heightened resurgent interest in studying the mechanisms of resistance and in developing new antibiotics. A comprehensive review has outlined the developments of tetracyclines prior to 1980 [47]. This review will highlight the pertinent advances in the tetracycline field during the last two decades, including recent progress on elucidating the mechanisms of resistance, and the development of novel tetracyclines to combat bacterial resistance. Most of the new tetracycline derivatives described in this review have been either prepared semisynthetically or isolated from fermentation. In the semisynthetic area, efflux inhibitors that are effective in an in vitro model have been identified. A new class of tetracyclines, named glycylcyclines has been the subject of numerous reports, and will be the major focus of this review. The glycylcyclines are currently the only derivatives that exhibit antibacterial activity comparable to that of the early tetracyclines when they were first introduced. These compounds show potent activity against a broad spectrum of Gram-positive and Gram-negative bacteria, including strains that carry the two major tetracycline-resistance determinants, efflux and ribosomal protection. Two of the glycylcycline derivatives. DMG-MINO and DMG-DMDOT, have been studied by several groups of investigators against a large number of clinical pathogens isolated from various sources. The spectrum of activity of these compounds includes organisms with resistance to antibiotics other than tetracyclines, e.g., methicillin-resistant staphylococci, penicillin-resistant streptococcus pneumoniae, and vancomycin-resistant enterococci. Their in vitro, as well as in vivo activity against bacteria with characterized tetracycline- or minocycline-resistant elements will be summarized. The structure-activity relationships of glycylcyclines and their mode of action will also be discussed.

Anti-Bacterial Agents↗

PubMed1997

Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC beta-lactamase, and the foss of an outer membrane protein.

Six Escherichia coli and 12 Klebsiella pneumoniae isolates from a single hospital expressed a common beta-lactamase with a pI of approximately 9.0 and were resistant to cefoxitin and cefotetan (MIC ranges, 64 to > 128 and 16 to > 128 micrograms/ml, respectively). Seventeen of the 18 strains produced multiple beta-lactamases. Most significantly, three K. pneumoniae strains were also resistant to imipenem (MICs, 8 to 32 micrograms/ml). Spectrophotometric beta-lactamase assays with purified enzyme indicated hydrolysis of cephamycins, in addition to cephaloridine and benzylpenicillin. The 4ene encoding the pI 9.0 beta-lactamase (designated ACT-1 for AmpC type) was cloned and sequenced, which revealed an ampC-type beta-lactamase gene that originated from Enterobacter cloacae and that had 86% sequence homology to the P99 beta-lactamase and 94% homology to the partial sequence of MIR-1. Southern blotting revealed that the gene encoding ACT-1 was on a large plasmid in some of the K. pneumoniae strains as well as on the chromosomes of all of the strains, suggesting that the gene is located on an easily mobilized element. Outer membrane protein profiles of the K. pneumoniae strains revealed that the three imipenem-resistant strains were lacking a major outer membrane protein of approximately 42 kDa which was present in the imipenem-susceptible strains. ACT-1 is the first plasmid-mediated AmpC-type beta-lactamase derived from Enterobacter which has been completely sequenced. This work demonstrates that in addition to resistance to cephamycins, imipenem resistance can occur in K. pneumoniae when a high level of the ACT-1 beta-lactamase is produced in combination with the loss of a major outer membrane protein.

Amino Acid Sequence↗

PubMed1995

The agr P2 operon: an autocatalytic sensory transduction system in Staphylococcus aureus.

The synthesis of virulence factors and other exoproteins in Staphylococcus aureus is controlled by the global regulator, agr. Expression of secreted proteins is up-regulated in the postexponential growth phase, whereas expression of surface proteins is down-regulated by agr. The agr locus consists of two divergent operons, transcribed from neighboring but non-overlapping promoters, P2 and P3. The P2 operon sequence, reported here, contains 4 open reading frames, agrA, C, D, and B, of which A and C appear to encode proteins of a classical 2-component signal transduction pathway. The P3 operon specifies a 0.5 kb transcript, RNA III, which is the actual effector of the agr response, and, incidentally, encodes the agr-regulated peptide delta-hemolysin. Transcriptional fusions have shown that both P2 and P3 are agr sensitive (function in an agr+ but not in an agr- background) and deletion analysis has shown that all four of the P2 ORFs are involved; agrA and agrC seem to be absolutely required for the transcriptional activation of the agr locus, whereas agrB and agrD seem to be partially required. Since transcription of P2 requires P2 operon products, the P2 operon is autocatalytic, and is thus admirably suited to the need for rapid production of exoproteins at a time when overall growth is coming to a halt.

Amino Acid Sequence↗

PubMed1995

Molecular epidemiology of Staphylococcus epidermidis blood isolates from neonatal intensive care unit patients.

Twelve episodes of Staphylococcus epidermidis bacteraemia occurred within three months in a neonatal intensive care unit. Plasmid profiles and Southern blot hybridization with five different probes were used to determine whether an endemic strain of S. epidermidis could be identified among the contemporary isolates. It was concluded that this methodology was satisfactory for differentiation between isolates of coagulase-negative staphylococci: fifteen isolates were divided in eight groups indicating that there was no single endemic strain causing the outbreak.

Bacteremia↗

PubMed1995

SHV-7, a novel cefotaxime-hydrolyzing beta-lactamase, identified in Escherichia coli isolates from hospitalized nursing home patients.

Four ceftazidime-resistant Escherichia coli strains were isolated from elderly nursing home patients in a New York hospital during 1993. Strains MCQ-2, MCQ-3, and MCQ-4 were determined to be identical by pulsed-field gel electrophoresis and plasmid profiles, whereas strain MCQ-1 was unique. Strain MCQ-1 was determined to produce a TEM-10 beta-lactamase. Strains MCQ-2, MCQ-3, and MCQ-4 were also noted to be resistant to cefotaxime. These three strains produced two beta-lactamases with pIs of 5.4 (TEM-1) and 7.6. beta-Lactamase assays revealed that the pI 7.6 enzyme hydrolyzed cefotaxime faster (at a relative hydrolysis rate of 30% compared with that of benzylpenicillin) than either ceftazidime or aztreonam (relative hydrolysis rates of 13 and 3.3%, respectively). Nucleotide sequencing of the gene encoding the pI 7.6 beta-lactamase from strain MCQ-3 revealed a blaSHV-type gene differing from the gene encoding SHV-1 at four nucleotides which resulted in amino acid substitutions: phenylalanine for isoleucine at position 8, serine for arginine at position 43, serine for glycine at position 238, and lysine for glutamate at position 240. This novel SHV-type extended-spectrum beta-lactamase is designated SHV-7.

Amino Acid Sequence↗