Balbina J. Plotkin

Lactones: generic inhibitors of enzymes?

The ability to affect eukaryotic and prokaryotic cellular growth, signaling and differentiation is a continuing focus in the pharmaceutical industry. The fundamental ability to affect these cellular processes is inherent in lactones. Lactones, which are ubiquitous in nature, reflect a broad phylogenetic diversity indicative of their ability to act as simple alkylating compounds, with their in situ activities falling into one of two categories, i.e., protect or conquer. Medically, their utility as pharmaceutical agents range from that of antimicrobial to anti-neoplastic agent depending on the functional groups attached.

Effect of androgens and glucocorticoids on microbial growth and antimicrobial susceptibility.

The effects of androgens, testosterone and dihydrotestosterone (DHT), of an environmental anti-androgen, 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (DDE), and of glucocorticoids, hydrocortisone and dexamethasone, on growth kinetics and antibiotic susceptibility of E. faecalis, E. coli, P. aeurginosa, and S. aureus were measured. For P. aeurginosa, the presence of either DHT or DDE caused at least a fourfold shift in the minimum inhibitory concentration (MIC) of cefepime and tobramycin. DHT and DDE also affected the response of E. faecalis to meropenem and norfloxacin, resulting in a shift from sensitive to intermediate resistance (four-fold increase in MIC). Hydrocortisone (2 μM) induced an increase in the sensitivity of S. aureus to erythromycin, as compared to hormone-free control (from 0.5 to 0.06 μg/mL). The susceptibility pattern of E. coli was unaffected by the hormones tested. These changes in susceptibility to antibiotics were unrelated to alterations in growth kinetics. For all organisms tested, the alterations in MICs occurred only in the presence of hormone, indicative of changes in the phenotype of these stable quality control strains.

Effect of insulin on microbial growth

The ability of insulin to affect the growth kinetics of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa was measured. For all organisms, insulin, in the absence of a metabolizable sugar source, i.e., glucose or starch in Mueller-Hinton medium, had no effect on generation time as compared with a homologous control. Response to insulin, in the form of increased or decreased generation times, for both Gram-positive and Gram-negative bacteria, was dependent on the concentration of insulin, the concentration of glucose present, and the initial concentration of bacteria exposed to the glucose and insulin.

Effect of Sub-MICs of Antibiotics on the Hydrophobicity and Production of Acidic Polysaccharide by Vibrio vulnificus

Background: Subinhibitory concentrations of antibiotics, which can occur in vivo, have been demonstrated to alter the production of bacterial virulence factors, including the capsule, or the interaction between microorganism and phagocyte by affecting surface hydrophobicity. Methods: Using a microtiter assay system, the effect of subinhibitory concentrations of amikacin, gentamicin, cephalothin and doxycycline on the surface hydrophobicity and production of acidic polysaccharide by Vibrio vulnificus (8 human isolates, 8 environmental isolates) was determined. Results: All four drugs, in a dose-dependent manner, caused alterations in adherence to polystyrene, a measure of surface hydrophobicity, and the production of acidic polysaccharides, as determined by Alcian blue staining. Conclusion: The changes in capsule production and surface hydrophobicity measured in response to sub-MICs of antibiotics appear to be independent variables.

C4-Alkylthiols with activity against Moraxella catarrhalis and Mycobacterium tuberculosis

Antimicrobial resistance represents a global threat to healthcare. The ability to adequately treat infectious diseases is increasingly under siege due to the emergence of drug-resistant microorganisms. New approaches to drug development are especially needed to target organisms that exhibit broad antibiotic resistance due to expression of β-lactamases which is the most common mechanism by which bacteria become resistant to β-lactam antibiotics. We designed and synthesized 20 novel monocyclic β-lactams with alkyl- and aryl-thio moieties at C4, and subsequently tested these for antibacterial activity. These compounds demonstrated intrinsic activity against serine β-lactamase producing Mycobacterium tuberculosis wild type strain (Mtb) and multiple (n=6) β-lactamase producing Moraxella catarrhalis clinical isolates.

Possible role of sarA in dehydroepiandrosterone-mediated increase in Staphylococcus aureus resistance to vancomycin.

Background: Dehydroepiandrosterone (DHEA), a steroid present throughout life, can induce an increase in resistance to vancomycin in methicillin-sensitive and methicillin-resistant clinical isolates of Staphylococcus aureus. Methods: The in vitro effect of DHEA on vancomycin killing of S. aureus with mutations in sarA and/or agr was determined by standard microtiter protocols and time to kill determinations. Results: Of the isolates tested, the strain with a deletion in sarAderived from a DHEA- responsive parent was not protected from vancomycin killing by DHEA. However, DHEA significantly (p < 0.01) slowed the rate of vancomycin killing of sarA–. Conclusion: These data indicate that sarA may play a role in DHEA-mediated protection from vancomycin killing of S. aureus.

Non-transpeptidase binding arylthioether β-lactams active against Mycobacterium tuberculosis and Moraxella catarrhalis.

The prevalence of drug resistance in both clinical and community settings as a consequence of alterations of biosynthetic pathways, enzymes or cell wall architecture is a persistent threat to human health. We have designed, synthesized, and tested a novel class of non-transpeptidase, β-lactamase resistant monocyclic β-lactams that carry an arylthio group at C4. These thioethers exhibit inhibitory and cidal activity against serine β-lactamase producing Mycobacterium tuberculosis wild type strain (Mtb) and multiple (n=8) β-lactamase producing Moraxella catarrhalis clinical isolates.

A method for the long-term cultivation of mammalian cells in the absence of oxygen: Characterization of cell replication, hypoxia-inducible factor expression and reactive oxygen species production

The center of tumors, stem cell niches and mucosal surfaces all represent areas of the body that are reported to be anoxic. However, long-term study of anoxic cell physiology is hindered by the lack of a sustainable method permitting cell cultivation in the complete absence of oxygen. A novel methodology was developed that enabled anoxic cell cultivation (17d maximum time tested) and cell passage. In the absence of oxygen, cell morphology is significantly altered. All cells tested exhibited morphologic changes, i.e., a combination of tethered (monolayer-like) and runagate (suspension-like) morphologies. Both morphologies replicated (Vero and HeLa cells tested) and could be passaged anaerobically. In the absence of exogenous oxygen, anoxic cells produced reactive oxygen species (ROS). Anaerobic runagate HeLa and Vero cells increased ROS production from day 3 to day 10 by 2- and 3-fold, respectively. In contrast, anoxic tethered HeLa and Vero cells either showed no significant change in ROS production between days 3 and 10 or exhibited a 3-fold decrease in ROS, respectively. Detection of ROS was inversely related to detection of hypoxia-inducible factor-1α (HIF1) mRNA and HIF-1 protein expression which cycled over a 10-day period. This methodology has broad applications for the study of tumor and stem cell physiology as well as gastrointestinal cell-microbiome interactions. In addition, sustainable anaerobic cell culture may lead to the identification of novel pathways and targets for chemotherapeutic drug development.

Attenuation of antimicrobial activity by the human steroid hormones

HighlightsDHEA (dehydroepiandosterone) increased Staphylococcus aureus resistance &bgr;‐1 defensin.DHEA affects the activity of positively‐charged antibiotics, including vancomycin.The effect of DHEA on S. aureus is phenotypic.DHEA altered surface charge, hydrophobicity, capsule and carotenoid production. Abstract Upon entering the human host, Staphylococcus aureus is exposed to endogenous steroid hormones. The interaction between S. aureus and dehydroepiandosterone (DHEA) results in an increased resistance to the host cationic defense peptide, &bgr;‐1 defensin, as well as vancomycin and other antibiotics that have a positive charge. The increased resistance to vancomycin is phenotypic and appears to correlate with a DHEA‐mediated alteration in cell surface architecture. DHEA‐mediated cell surface changes include alterations in: cell surface charge, surface hydrophobicity, capsule production, and carotenoid production. In addition, exposure to DHEA results in decreased resistance to lysis by Triton X‐100 and lysozyme, indicating activation of murien hydrolase activity. We propose that DHEA is an interspecies quorum‐like signal that triggers innate phenotypic host survival strategies in S. aureus that include increased carotenoid production and increased vancomycin resistance. Furthermore, this DHEA‐mediated survival system may share the cholesterol‐squalene pathway shown to be statin sensitive thus, providing a potential pathway for drug targeting.

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi.

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSVs effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.